Process for producing glycine

ABSTRACT

Disclosed is a method for producing glycine, which comprises subjecting an aqueous solution of glycinonitrile to a hydrolysis reaction in a hydrolysis reaction system under the action of a microbial enzyme, thereby converting the glycinonitrile to glycine while by-producing ammonia, wherein the hydrolysis reaction system contains at least one organic impurity compound inhibiting the microbial enzyme, wherein the organic impurity compound has a molecular weight of 95 or more and contains at least one member selected from the group consisting of a nitrile group, a carboxyl group, an amide group, an amino group, a hydroxyl group and a trimethyleneamine structure, and wherein the hydrolysis reaction is performed under conditions wherein, during the hydrolysis reaction, the content of the organic impurity compound inhibiting the microbial enzyme in the hydrolysis reaction system is maintained at a level of 10% by weight or less, based on the weight of the hydrolysis reaction system.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for producing glycine.More particularly, the present invention is concerned with a method forproducing glycine, which comprises subjecting an aqueous solution ofglycinonitrile to a hydrolysis reaction in a hydrolysis reaction systemunder the action of a microbial enzyme, thereby converting theglycinonitrile to glycine while by-producing ammonia, wherein thehydrolysis reaction system contains at least one organic impuritycompound inhibiting the microbial enzyme, wherein the organic impuritycompound has a molecular weight of 95 or more and contains a specificstructure, and wherein the hydrolysis reaction is performed underconditions wherein, during the hydrolysis reaction, the content of theorganic impurity compound inhibiting the microbial enzyme in thehydrolysis reaction system is maintained at a level of 10% by weight orless, based on the weight of the hydrolysis reaction system. By the useof the method of the present invention, a high purity glycine which isuseful as a food additive and as a raw material for synthesizingpharmaceuticals, agricultural chemicals and detergents can be producedeasily and efficiently on a commercial scale without causing a heavyburden on the environment.

[0003] 2. Prior Art

[0004] Conventionally, glycine has been produced by a process whichcomprises: synthesizing glycinonitrile from formaldehyde, hydrogencyanide and ammonia by the Strecker method; converting the synthesizedglycinonitrile into glycine soda and ammonia by hydrolysis using analkali (such as caustic soda); neutralizing the glycine soda with anacid (such as sulfuric acid) to obtain glycine; and recovering theglycine by crystal-deposition (see Unexamined Japanese PatentApplication Laid-Open Specification Nos. Sho 43-29929, Sho 51-19719, Sho49-14420 and Sho 49-35329). As apparent from the above, such aconventional hydrolysis method employing a base uses an alkali and anacid in amounts each equivalent to the amount of glycine produced.Therefore, such method has a problem in that large amounts of salts areby-produced and the disposal of the by-products causes a heavy burden onthe environment. Further, since the solubilities of the by-producedsalts are similar to that of glycine, the recovery of glycine cannot beachieved by a single-step crystal-deposition and, therefore, acumbersome operation, such as repetition of a cycle comprisingcrystal-deposition and circulation of a mother liquor, is necessary forrecovering glycine (see Unexamined Japanese Patent Application Laid-OpenSpecification No. Sho 51-34113 (corresponding to DE 2541677-B and NL7511023-B)). In addition, an aqueous glycinonitrile solution as anintermediate is unstable at a pH of 2.5 or more, and it is known thatthe higher the temperature, the greater the likelihood thatglycinonitrile suffers denaturation, such as decomposition anddiscoloration (see Unexamined Japanese Patent Application Laid-OpenSpecification Nos. Sho 49-14420, Sho 54-46720 and Sho 54-46721). In“Kogyo Kagaku Zasshi (Journal of Industrial Chemistry)”, Volume 70, page54 (1967) published by Japanese Chemical Society, Japan, it is statedthat hydrogen cyanide is likely to be denatured by polymerization underalkaline conditions, and black solids are generated as thepolymerization proceeds. Further, “Jikken Kagaku Kouza (Lectures onExperimental Chemistry)”, 1st ed., page 347, published by JapaneseChemical Society, Japan, describes that a cyanomethyl group ofglycinonitrile and the like is likely to be denatured by additionpolymerization under alkaline conditions, resulting in the generation ofpyridine compounds and pyrimidine compounds. Unexamined Japanese PatentApplication Laid-Open Specification No. Sho 62-212357 (corresponding toU.S. Pat. No. 4,661,614) discloses that an imine compound, such asiminodiacetonitrile, can be synthesized from formaldehyde, hydrogencyanide and ammonia. Examined Japanese Patent Application PublicationSpecification No. Sho 51-244815 discloses that when glycinonitrile isheated, glycinonitrile generates ammonia and imine compounds (such asiminodiacetonitrile), and further heating causes the imine compounds todenature, resulting in the generation of black compounds. Therefore, inthe conventional methods, a lowering of the yield of glycine due to theabove-mentioned decomposition and denaturation is unavoidable. Further,the conventional methods have a defect in that a cumbersome treatmentemploying an activated carbon or a special ion exchange resin isnecessary for removing a discolored matter (see Unexamined JapanesePatent Application Laid-Open Specification No. Hei 3-190851 andUnexamined Japanese Patent Application Laid-Open Specification No. Hei4-226949 (corresponding to EP 459803-B)).

[0005] As a method for hydrolyzing glycinonitrile under moderateconditions without using large amounts of an alkali and an acid, thereis known a method in which glycinonitrile is simply hydrolyzed using amicroorganism having the activity to hydrolyze a nitrile group, therebyobtaining glycine and ammonium. Examined Japanese Patent ApplicationPublication Specification No. Sho 58-15120 (corresponding to FrenchPatent No. 225585) discloses a method in which a hydrolysis reaction isconducted using Brevibacterium R312 suspended in a reaction mediumliquid which has been adjusted to have a pH value of 8 with causticpotash or the like. Unexamined Japanese Patent Application Laid-OpenSpecification No. Hei 3-62391 (corresponding to EP 187680-B) discloses amethod in which a hydrolysis reaction is conducted using CorynebacteriumN-774 suspended in a reaction medium liquid which is a phosphate bufferhaving a pH value of 7.7. Unexamined Japanese Patent ApplicationLaid-Open Specification No. Hei 3-280889 (corresponding to EP 450885-B)discloses a method in which glycine is produced from glycinonitrile byusing a microorganism belonging to the genus Rhodococcus, Arthrobacter,Caseobacter, Pseudomonas, Enterobacter, Acinetobacter, Alkaligenes,Corynebacterium or Streptomyces which is capable of hydrolyzing anitrile group, wherein the microorganism is suspended in a reactionmedium liquid which is a phosphate buffer having a pH value of 7.7.However, as shown in the working examples of these patent documents,these methods require that a lyophilized microorganism be used in anamount which is equal to or greater than the amount of glycine, oralternatively, the reaction be performed for 30 hours using a largeamount of a lyophilized microorganism, namely 5% by weight or more,based on the weight of glycine. Further, as also shown in the workingexamples of these patent documents, these methods require that aneutralizing agent be successively added to the reaction system tomaintain the pH of the reaction system within the neutral range so as tomaintain the activity of the microorganism. In general, for neutralizingan ammonium salt of glycine, sulfuric acid or phosphoric acid is addedto the reaction system, and hence a large amount of ammonium sulfate orammonium phosphate is likely to remain in the reaction system.Therefore, the above-mentioned methods for producing glycine by using amicroorganism are disadvantageous in that a large amount of an acid mustbe used and a large amount of waste must be discarded. Further, theabove-mentioned methods using a microorganism have the followingproblems. In the above-mentioned methods using a microorganism, theoperation for recovering glycine needs a step of adding methanol or thelike in addition to the concentration step, so that the operation forrecovering glycine becomes complicated, as compared to the case of thealkaline hydrolysis method mentioned above (see Examined Japanese PatentApplication Publication Specification No. Sho 58-15120 (corresponding toFrench Patent No. 225585)). Further, the above-mentioned methods use alarge amount of a microorganism, and this results in a further increasein the amount of waste. On the other hand, a method is known whichemploys a microorganism and electric dialysis and in which glycine andammonia are separately recovered while recycling an alkali (seeUnexamined Japanese Patent Application Laid-Open Specification No. Sho10-179183 (corresponding to U.S. Pat. No. 5,932,454 and EP 852261-A)).This method is a method for producing glycine, comprising the followingsteps: a step of producing ammonium salts of organic acids (includingglycine) by using a microorganism; a step of converting the ammoniumsalts into alkali salts, thereby eliminating ammonia; a step ofrecovering the eliminated ammonia; a step of separating the alkali andthe organic acids from each other by electric dialysis; a step ofextracting the organic acids with an organic solvent; and a step ofseparating the organic acids from the organic solvent. In the workingexamples of the above-mentioned patent document, only 0.3 mole of anorganic acid is obtained from the microorganism cultured in 20 g ofglycerin medium, showing that the activity of the microorganism is verylow. Thus, this method has disadvantages in that multiple steps andcumbersome operations are necessary, a large amount of electricity isconsumed, and a large amount of a microorganism is used and discarded.

[0006] As apparent from the above, the conventional methods forproducing glycine from glycinonitrile by the use of a microorganism havedisadvantages in that both the activity per unit amount of a lyophilizedmicroorganism and the activity per unit time are low, and large amountsof culture medium and microorganism must be discarded. In addition, inthe case of a method in which the electric dialysis is not employed,there are problems in that the recovery of ammonia is difficult due tothe use of a neutralizing agent for adjusting the pH of the reactionsystem or for recovering glycine, and that a step of removing theneutralizing agent is necessary. Even in the case of a method in whichthe electric dialysis is employed, the recovery of ammonia requires notonly electricity but also a cumbersome, multiple-step operation.Therefore, the commercial practice of this method cannot besatisfactorily performed.

SUMMARY OF THE INVENTION

[0007] In this situation, the present inventors have made extensive andintensive studies for finding a reaction method and reaction conditionswhich can solve the above-mentioned problems, and for findingmicroorganisms which are suitable for use in such a reaction method. Asa result, it has unexpectedly been found that the above-mentionedobjective can be attained by a method for producing glycine, whichcomprises: subjecting an aqueous solution of glycinonitrile to ahydrolysis reaction in a hydrolysis reaction system under the action ofa microbial enzyme, thereby converting the glycinonitrile to glycinewhile by-producing ammonia, wherein the hydrolysis reaction systemcontains at least one organic impurity compound inhibiting the microbialenzyme, wherein the organic impurity compound has a molecular weight of95 or more and contains a specific structure, and wherein the hydrolysisreaction is performed under conditions wherein, during the hydrolysisreaction, the content of the organic impurity compound inhibiting themicrobial enzyme in the hydrolysis reaction system is maintained at alevel of 10% by weight or less, based on the weight of the hydrolysisreaction system. That is, it has surprisingly been found that by the useof the above-mentioned method, advantages are achieved only in thatglycine can be produced without the need to use and discard largeamounts of a microorganism, a culture medium, an acid, an alkali and thelike, but also in that the discoloration of glycine can be suppressed,and both the glycine production activity per unit weight of amicroorganism and the glycine production activity per unit time becomehigh, and both glycine and ammonia can be stoichiometrically producedwithout decomposition or consumption thereof and can be separately andeasily recovered. The present invention has been completed, based on theabove-mentioned novel findings.

[0008] Accordingly, it is a primary object of the present invention toprovide a method for producing glycine, which is advantageous not onlyin that high purity glycine and high purity ammonia can be producedefficiently and stoichiometrically and can be separately recovered, butalso in that the method does not cause a heavy burden on theenvironment.

[0009] The foregoing and other objects, features and advantages of thepresent invention will be apparent to those skilled in the art from thefollowing detailed description and the appended claims taken inconnection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

[0010] In the drawing:

[0011]FIG. 1 is a diagram showing the production system used in Example12 for producing glycine.

[0012] Description of Reference Numerals 1: aqueous formaldehydesolution 2: liquid hydrogen cyanide 3: sodium hydroxide 4: gaseousammonia 5: microbial suspension 6,7,8: autoclave 9: flash distillationapparatus 10: liquid ammonia 11,16: intermediate tank 12: hydrolysisreactor 13: continuous centrifuge 14: microorganism to be discarded 15:circulation type ultrafiltration apparatus 17: activated carbon column18: continuous crystal-deposition apparatus 19: water which has beenevaporated and recovered 20: blow 21: filtrate 22: glycine crystals

DETAILED DESCRIPTION OF THE INVENTION

[0013] In one aspect of the present invention, there is provided amethod for producing glycine, comprising:

[0014] providing glycinonitrile in the form of an aqueous solutionthereof,

[0015] subjecting the aqueous solution of glycinonitrile to a hydrolysisreaction in a hydrolysis reaction system under the action of a microbialenzyme having the activity to hydrolyze a nitrile group, therebyconverting the glycinonitrile to glycine while by-producing ammonia,

[0016] the hydrolysis reaction system containing at least one organicimpurity compound inhibiting the microbial enzyme, wherein the at leastone organic impurity compound inhibiting the microbial enzyme has amolecular weight of 95 or more and contains at least one member selectedfrom the group consisting of a nitrile group, a carboxyl group, an amidegroup, an amino group, a hydroxyl group and a trimethyleneaminestructure, wherein the trimethyleneamine structure has a skeletonrepresented by the following formula (1):

[0017] wherein n represents an integer of 1 or more,

[0018] the hydrolysis reaction being performed under conditions wherein,during the hydrolysis reaction, the content of the organic impuritycompound inhibiting the microbial enzyme in the hydrolysis reactionsystem is maintained at a level of 10% by weight or less, based on theweight of the hydrolysis reaction system, and

[0019] isolating the glycine from the hydrolysis reaction system.

[0020] For easy understanding of the present invention, the essentialfeatures and various preferred embodiments of the present invention areenumerated below.

[0021] 1. A method for producing glycine, comprising:

[0022] providing glycinonitrile in the form of an aqueous solutionthereof,

[0023] subjecting the aqueous solution of glycinonitrile to a hydrolysisreaction in a hydrolysis reaction system under the action of a microbialenzyme having the activity to hydrolyze a nitrile group, therebyconverting the glycinonitrile to glycine while by-producing ammonia,

[0024] the hydrolysis reaction system containing at least one organicimpurity compound inhibiting the microbial enzyme, wherein the at leastone organic impurity compound inhibiting the microbial enzyme has amolecular weight of 95 or more and contains at least one member selectedfrom the group consisting of a nitrile group, a carboxyl group, an amidegroup, an amino group, a hydroxyl group and a trimethyleneaminestructure, wherein the trimethyleneamine structure has a skeletonrepresented by the following formula (1):

[0025] wherein n represents an integer of 1 or more,

[0026] the hydrolysis reaction being performed under conditions wherein,during the hydrolysis reaction, the content of the organic impuritycompound inhibiting the microbial enzyme in the hydrolysis reactionsystem is maintained at a level of 10% by weight or less, based on theweight of the hydrolysis reaction system, and

[0027] isolating the glycine from the hydrolysis reaction system.

[0028] 2. The method according to item 1 above, wherein the at least oneorganic impurity compound inhibiting the microbial enzyme is produced asa by-product in at least one reaction selected from the group consistingof the synthesis of glycinonitrile from hydrogen cyanide, formaldehydeand ammonia, and the hydrolysis of the glycinonitrile into glycine andammonia.

[0029] 3. The method according to item 1 or 2 above, wherein the atleast one organic impurity compound inhibiting the microbial enzymecomprises a compound represented by the following formula (2):

NH_(3-n)(CH₂Y¹)_(n)  (2)

[0030] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; and n represents an integer of 2 or 3.

[0031] 4. The method according to item 1 or 2, wherein the at least oneorganic impurity compound inhibiting the microbial enzyme comprises atleast one compound selected from the group consisting of the followingcompounds (a) and (b):

[0032] (a) a compound represented by the following formula (3):

[0033] wherein:

[0034] Y¹ represents a nitrile group, a carboxyl group or an amidegroup;

[0035] each Y² independently represents an amino group or a hydroxylgroup;

[0036] n represents an integer of 0 or more; and

[0037] the or each Z is independently represented by the followingformula (4) or (5):

[0038] wherein each Y independently represents an amino group or ahydroxyl group, and

[0039] (b) a compound represented by the following formula (6) or (7):

[0040] wherein each Y² independently represents an amino group or ahydroxyl group; and each Z² is independently represented by thefollowing formula (8) or (9):

[0041] or

Z¹ _(n)—H  (9)

[0042] wherein:

[0043] Y² represents an amino group or a hydroxyl group;

[0044] the or each Z is as defined for formula (3); and

[0045] n represents an integer of 0 or more.

[0046] 5. The method according to item 1 or 2 above, wherein the atleast one organic impurity compound inhibiting the microbial enzymecomprises at least one compound selected from the group consisting ofthe following compounds (c) and (d):

[0047] (c) a compound represented by the following formula (10) or (11):

[0048] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group,

[0049] and

[0050] (d) a compound represented by the following formula (12):

(HCN)_(n)  (12)

[0051] wherein n represents an integer of 4 or more.

[0052] 6. The method according to item 1 or 2 above, wherein the atleast one organic impurity compound inhibiting the microbial enzymecomprises a compound having in a molecule thereof at least one skeletonselected from the group consisting of the following skeletons (e) and(f):

[0053] (e) a skeleton represented by the following formula (13):

[0054] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; and n represents an integer of 2 ormore, and

[0055] (f) a skeleton represented by the following formula (14) or (15):

[0056] wherein:

[0057] the or each Y independently represents a nitrile group, acarboxyl group or an amide group;

[0058] each Y² independently represents an amino group or a hydroxylgroup; and

[0059] n represents an integer of 1 or more.

[0060] 7. The method according to item 1 or 2 above, wherein the atleast one organic impurity compound inhibiting the microbial enzymecomprises a compound having in a molecule thereof at least one skeletonselected from the group consisting of the following skeletons (g) and(h):

[0061] (g) a skeleton represented by the following formula (16):

[0062] wherein:

[0063] each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group;

[0064] each Y² independently represents an amino group or a hydroxylgroup;

[0065] and n represents an integer of 2 or more, and

[0066] (h) a skeleton represented by the following formula (17) or (18):

[0067] wherein:

[0068] the or each Y¹ independently represents a nitrile group, acarboxyl group or an amide group;

[0069] each Y² independently represents an amino group or a hydroxylgroup; and

[0070] n represents an integer of 1 or more.

[0071] 8. The method according to item 1 or 2 above, wherein the atleast one organic impurity compound inhibiting the microbial enzymecomprises hexamethylenetetramine.

[0072] 9. The method according to any one of items 1 to 8 above, whereinthe at least one organic impurity compound inhibiting the microbialenzyme exhibits a peak between 53 ppm and 100 ppm in a ¹³C-NMR spectrumas measured in heavy water.

[0073] 10. The method according to any one of items 1 to 9 above,wherein the at least one organic impurity compound inhibiting themicrobial enzyme exhibits absorption maximums between 340 nm and 380 nmand between 440 nm and 480 nm in an ultraviolet-visible absorptionspectrum as measured with respect to the hydrolysis reaction system.

[0074] 11. The method according to any one of items 1 to 10 above,wherein the at least one organic impurity compound inhibiting themicrobial enzyme has a molecular weight of 130 or more.

[0075] 12. The method according to any one of items 1 to 11 above,wherein the amount of the at least one organic impurity compoundinhibiting the microbial enzyme is 1% by weight or less, based on theweight of the hydrolysis reaction system.

[0076] 13. The method according to any one of items 1 to 12 above,wherein the hydrolysis reaction system has oxygen dissolved therein inan amount of 5 ppm by weight or less, based on the weight of thehydrolysis reaction system.

[0077] 14. The method according to any one of items 1 to 13 above,wherein the hydrolysis is conducted using a closed reaction system, areaction system which is pressurized with an inert gas, a reactionsystem through which an inert gas is flowed or a reaction system whichis under a pressure of less than atmospheric pressure, so that theamount of oxygen dissolved in the hydrolysis reaction system issuppressed.

[0078] 15. The method according to any one of items 1 to 14 above,wherein the hydrolysis is conducted in the hydrolysis reaction systemhaving ammonia dissolved therein.

[0079] 16. The method according to any one of items 1 to 15 above,wherein the hydrolysis is conducted in the hydrolysis reaction systemcontaining an electrolyte in an amount of 2% by weight or less, based onthe weight of the glycinonitrile.

[0080] 17. The method according to any one of items 1 to 16 above,wherein the microbial enzyme having the activity to hydrolyze a nitrilegroup is derived from a microorganism belonging to a genus selected fromthe group consisting of Acinetobacter, Rhodococcus, Corynebacterium,Alcaligenes, Mycobacterium, Rhodopseudomonas and Candida.

[0081] 18. The method according to item 17 above, wherein the microbialstrain of the Acinetobacter is Acinetobacter sp. AK226, deposited withthe National Institute of Bioscience and Human-Technology, Japan underthe accession number FERM BP-2451, or Acinetobacter sp. AK227, depositedwith the National Institute of Bioscience and Human-Technology, Japanunder the accession number FERM BP-7413.

[0082] 19. The method according to item 17 above, wherein the microbialstrain of the Rhodococcus is Rhodococcus maris BP-479-9, deposited withthe National Institute of Bioscience and Human-Technology, Japan underthe accession number FERM BP-5219.

[0083] 20. The method according to item 17 above, wherein the microbialstrain of the Corynebacterium is Corynebacterium sp. C5, deposited withthe National Institute of Bioscience and Human-Technology, Japan underthe accession number FERM BP-7414, or Corynebacterium nitrilophilus,deposited with the American Type Culture Collection, U.S.A under theaccession number ATCC 21419.

[0084] 21. The method according to item 17 above, wherein the microbialstrain of the Alcaligenes is Alcaligenes faecalis IFO 13111, depositedwith the National Institute of Bioscience and Human-Technology, Japanunder the accession number FERM BP-4750.

[0085] 22. The method according to item 17 above, wherein the microbialstrain of the Mycobacterium is Mycobacterium sp. AC777, deposited withthe National Institute of Bioscience and Human-Technology, Japan underthe accession number FERM BP-2352.

[0086] 23. The method according to item 17 above, wherein the microbialstrain of the Rhodopseudomonas is Rhodopseudomonas spheroides, depositedwith the American Type Culture Collection, U.S.A under the accessionnumber ATCC 11167.

[0087] 24. The method according to item 17 above, wherein the microbialstrain of the Candida is Candida tropicalis, deposited with the AmericanType Culture Collection, U.S.A under the accession number ATCC 20311.

[0088] 25. The method according to any one of items 1 to 24 above,wherein the isolation of the glycine from the hydrolysis reaction systemis conducted while recovering the by-produced ammonia separately fromthe recovery of glycine.

[0089] 26. The method according to item 25 above, wherein the glycineand the ammonia are separately recovered by at least one operationselected from the group consisting of distillation, reactivedistillation, entrainment by inert gas, ion exchange, extraction,reprecipitation using a poor solvent, and crystal-deposition byconcentration or cooling.

[0090] 27. The method according to item 26 above, wherein the ammonia isrecovered by distillation, reactive distillation or entrainment by aninert gas, and the glycine is recovered by subjecting a liquid remainingafter the recovery of the ammonia to crystal-deposition by concentrationor cooling.

[0091] 28. The method according to any one of items 1 to 27 above, whichcomprises:

[0092] (1) reacting hydrogen cyanide with formaldehyde in an aqueousmedium in the presence of an alkali catalyst in a closed reaction systemto obtain glycolonitrile in the form of an aqueous solution thereof,

[0093] (2) adding ammonia to the aqueous solution of glycolonitrile toeffect a reaction between the glycolonitrile and the ammonia, to therebyobtain glycinonitrile in the form of an aqueous solution thereof whileproducing water,

[0094] (3) separating most of the ammonia and a part of the water fromthe obtained aqueous solution of glycinonitrile by distillation tothereby obtain a hydrolysis reaction system containing theglycinonitrile in the form of an aqueous solution thereof and theammonia remaining unseparated, wherein the separated ammonia isrecovered for recycle thereof to step (2),

[0095] (4) subjecting the hydrolysis reaction system to a hydrolysisreaction under the action of a microbial enzyme produced by amicroorganism added in the hydrolysis reaction system which is in aclosed system, thereby converting the glycinonitrile to glycine whileby-producing ammonia,

[0096] (5) separating the microorganism and the microbial enzyme by atleast one operation selected from the group consisting of centrifugalfiltration and membrane filtration, wherein the microorganism and themicrobial enzyme are recovered for recycle thereof to step (4),

[0097] (6) separating a part of organic impurity compounds inhibitingthe microbial enzyme which compounds are by-produced in steps (1) to (5)by at least one operation selected from the group consisting of membranefiltration and adsorbent-separation,

[0098] (7) separating by distillation the ammonia by-produced in step(4) and an excess amount of water which remains in the hydrolysisreaction system after step (4), wherein the separated ammonia isrecovered for recycle thereof to step (2),

[0099] (8) after or simultaneously with step (7), separating the glycineby crystal-deposition, and

[0100] (9) drying the crystals of the glycine.

[0101] 29. A method for producing glycine, comprising providingglycinonitrile in the form of an aqueous solution thereof, subjectingthe aqueous solution of glycinonitrile to a hydrolysis reaction, therebyconverting the glycinonitrile to glycine while by-producing ammonia, andisolating the glycine from the hydrolysis reaction system, wherein thehydrolysis of glycinonitrile is conducted in the presence of ammonia.

[0102] 30. The method according to item 29 above, wherein the amount ofthe ammonia is from 0.001 to 5 mol, relative to 1 mole of theglycinonitrile.

[0103] 31. A method for producing glycine, comprising providingglycinonitrile in the form of an aqueous solution thereof, subjectingthe aqueous solution of glycinonitrile to a hydrolysis reaction, therebyconverting the glycinonitrile to glycine while by-producing ammonia, andisolating the glycine from the hydrolysis reaction system, wherein theisolation of the glycine from the hydrolysis reaction system isconducted while recovering the by-produced ammonia separately from therecovery of glycine in the absence of a base and an acid.

[0104] 32. The method according to item 31 above, wherein the glycineand ammonia are separately recovered by at least one operation selectedfrom the group consisting of distillation, reactive distillation,entrainment by an inert gas, ion exchange, extraction, reprecipitationusing a poor solvent, and crystal-deposition by concentration orcooling.

[0105] 33. The method according to item 32 above, wherein the ammonia isrecovered by distillation, reactive distillation or entrainment by aninert gas, and the glycine is recovered by subjecting a liquid remainingafter the recovery of the ammonia to crystal-deposition by concentrationor cooling.

[0106] Hereinbelow, the present invention will be described in detail.

[0107] In the present invention, it is preferred that glycinonitrile issynthesized from hydrogen cyanide, formaldehyde and ammonia.Glycinonitrile can be synthesized by the conventional methods.Specifically, glycinonitrile can be synthesized by, for example, amethod in which glycolonitrile (hydroxyacetonitrile) is synthesized fromhydrogen cyanide and formaldehyde and then ammonia is added to andreacted with the glycolonitrile to thereby obtain glycinonitrile; or amethod in which glycinonitrile is directly synthesized from hydrogencyanide, formaldehyde and ammonia.

[0108] The hydrolysis reaction system used in the present inventioncomprises glycolonitrile, glycinonitrile, glycine, ammonia, water, acatalyst, an organic impurity compound inhibiting a microbial enzyme andthe like.

[0109] The organic impurity compound inhibiting the microbial enzymewhich is contained in the hydrolysis reaction system is either acompound which inhibits a microbial enzyme reversibly or a compoundwhich inhibits a microbial enzyme irreversibly to deactivate the enzymeand make it impossible to reuse the microbial enzyme. Such organicimpurity compound has a molecular weight of 95 or more and contains atleast one member selected from the group consisting of a nitrile group,a carboxyl group, an amide group, an amino group, a hydroxyl group and atrimethyleneamine structure. For example, the organic impurity compoundmay be any of the following substances: raw materials used for producingglycine; additives contained in a catalyst and the like; and theimpurities contained in the raw materials. In addition, the organicimpurity compound may be any of the following substances: the compoundswhich are by-produced during the synthesis of glycinonitrile fromhydrogen cyanide, formaldehyde and ammonia; and the compounds which areby-produced during the hydrolysis of glycinonitrile (aminoacetonitrile)into glycine and ammonia. Further, the organic impurity compound may beany of the following substances: the compounds which are by-producedduring the production of the microbial enzyme having the activity tohydrolyze a nitrile group; and the by-products which are recycled fromthe step of separating and purifying glycine.

[0110] Specific examples of organic impurity compounds, which arecontained in the hydrolysis reaction system used in the presentinvention, include at least one compound selected from the groupconsisting of the following compounds (A) to (F).

[0111] (A) A compound represented by the following formula (2):

NH_(3-n)(CH₂Y¹)_(n)  (2)

[0112] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; and n represents an integer of 2 or 3.

[0113] Compounds represented by formula (2) above are a condensationcompound containing an imine (—NH—) structure or a nitrilo (—N═)structure and a compound formed by hydrolysis thereof. As examples ofsuch condensation compounds, there can be mentioned iminodiacetonitrileand nitrilotriacetonitrile which are known to be formed by thecondensation reaction between glycolonitrile and glycinonitrile.Examples of compounds formed by the hydrolysis of the nitrile group ofsuch condensation compounds include iminodiacetic acid,cyanomethylaminoacetic acid, iminodiacetoamide,cyanomethylaminoacetoamide, carbamoylmethylaminoacetic acid,nitrilotriacetic acid, N-(cyanomethyl)iminodiacetic acid,N,N-bis(cyanomethyl)aminoacetic acid, N-(carbamoylmethyl)iminodiaceticacid, N,N-bis(carbamoylmethyl)aminoacetic acid, nitrilotriacetoamide,N-(cyanomethyl)iminodiacetoamide, N,N-bis(cyanomethyl)aminoacetoamideand the like.

[0114] (B) At least one compound selected from the group consisting ofthe following compounds (a) and (b):

[0115] (a) a compound represented by the following formula (3):

[0116] wherein:

[0117] Y¹ represents a nitrile group, a carboxyl group or an amidegroup;

[0118] each Y² independently represents an amino group or a hydroxylgroup;

[0119] n represents an integer of 0 to 100; and

[0120] the or each Z¹ is independently represented by the followingformula (4) or (5):

[0121] wherein each Y independently represents an amino group or ahydroxyl group,

[0122] and

[0123] (b) a compound represented by the following formula (6) or (7):

[0124] wherein each Y² independently represents an amino group or ahydroxyl group; and each Z² is independently represented by thefollowing formula (8) or (9):

[0125] wherein:

[0126] Y² represents an amino group or a hydroxyl group;

[0127] the or each Z¹ is as defined for formula (3); and

[0128] n represents an integer of 0 to 100.

[0129] Compounds represented by formula (3) above are an additionpolymerization compound and a compound formed by the hydrolysis of thenitrile group of the addition polymerization compound. Compoundsrepresented by formula (6) are an addition cyclization compound having apyrimidine skeleton, which is formed by the cyclization of theabove-mentioned addition polymerization compound, and a compound formedby the hydrolysis of the nitrile group of such an addition cyclizationcompound. Compounds represented by formula (7) are an additioncyclization compound having a pyridine skeleton, which is formed by thecyclization of the above-mentioned addition polymerization compound, anda compound formed by the hydrolysis of the nitrile group of such anaddition cyclization compound. As exemplified by the addition reactionof phenylacetonitrile, which is described in “Jikken Kagaku Kouza(Lectures on Experimental Chemistry)”, 1st ed., Volume 18-2, page 347,published by Japanese Chemical Society, Japan, such addition compoundsare known to be produced by a mechanism by which a cyanomethyl groupundergoes an addition dimerization or addition trimerization, which isoptionally followed by cyclization or polymerization.

[0130] Examples of addition dimers represented by formula (3) aboveinclude compounds obtained by an addition reaction betweenglycolonitrile and glycinonitrile, namely1,3-diamino-2-imino-butylonitrile, 1,3-dihydroxy-2-imino-butylonitrile,1-amino-2-imino-3-hydroxybutylonitrile and3-amino-2-imino-1-hydroxybutylonitrile. Examples of compounds formed bythe hydrolysis of the nitrile group of the above-mentioned compoundsinclude 1,3-diamino-2-imino-butylic acid, 1,3-dihydroxy-2-imino-butylicacid, 1-amino-2-imino-3-hydroxybutylic acid,3-amino-2-imino-1-hydroxybutylic acid, 1,3-diamino-2-imino-butylamide,1,3-dihydroxy-2-imino-butylamide, 1-amino-2-imino-3-hydroxybutylamide,3-amino-2-imino-1-hydroxybutylamide and the like.

[0131] As examples of addition trimers which are represented by formula(3) above and which contain a structure represented by formula (4)above, i.e., a structure wherein a nitrile group is addition-bonded to amethylene group, there can be mentioned1,3,5-triamino-2,4-diiminohexanenitrile,1,3-diamino-2,4-diimino-5-hydroxyhexanenitrile,1,5-diamino-2,4-diimino-3-hydroxyhexanenitrile,3,5-diamino-2,4-diimino-1-hydroxyhexanenitrile,1-amino-2,4-diimino-3,5-dihydroxyhexanenitrile,3-amino-2,4-diimino-1,5-dihydroxyhexanenitrile,5-amino-2,4-diimino-1,3-dihydroxyhexanenitrile,2,4-diimino-1,3,5-trihydroxyhexanenitrile and the like. Examples ofcompounds formed by the hydrolysis of the nitrile group of theabove-mentioned compounds include 1,3,5-triamino-2,4-diiminohexanonicacid, 1,3-diamino-2,4-diimino-5-hydroxyhexanonic acid,1,5-diamino-2,4-diimino-3-hydroxyhexanonic acid,3,5-diamino-2,4-diimino-1-hydroxyhexanonic acid,1-amino-2,4-diimino-3,5-dihydroxyhexanonic acid,3-amino-2,4-diimino-1,5-dihydroxyhexanonic acid,5-amino-2,4-diimino-1,3-dihydroxyhexanonic acid,2,4-diimino-1,3,5-trihydroxyhexanonic acid,1,3,5-triamino-2,4-diiminohexaneamide,1,3-diamino-2,4-diimino-5-hydroxyhexaneamide,1,5-diamino-2,4-diimino-3-hydroxyhexaneamide,3,5-diamino-2,4-diimino-1-hydroxyhexaneamide,1-amino-2,4-diimino-3,5-dihydroxyhexaneamide,3-amino-2,4-diimino-1,5-dihydroxyhexaneamide,5-amino-2,4-diimino-1,3-dihydroxyhexaneamide,2,4-diimino-1,3,5-trihydroxyhexaneamide and the like.

[0132] As examples of addition trimers which are represented by formula(3) above and which contain a structure represented by formula (5)above, i.e., a structure wherein a nitrile group is addition-bonded toan imino group, there can be mentioned2,4-diamino-3-(2-amino-1-iminoethylimino)butylonitrile,2-amino-4-hydroxy-3-(2-amino-1-iminoethylimino)butylonitrile,4-amino-2-hydroxy-3-(2-amino-1-iminoethylimino)butylonitrile,2,4-dihydroxy-3-(2-amino-1-iminoethylimino)butylonitrile,2,4-diamino-3-(2-hydroxy-1-iminoethylimino)butylonitrile,2-amino-4-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylonitrile,4-amino-2-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylonitrile,2,4-dihydroxy-3-(2-hydroxy-1-iminoethylimino)butylonitrile and the like.Examples of compounds formed by the hydrolysis of the nitrile group ofthe above-mentioned compounds include2,4-diamino-3-(2-amino-1-iminoethylimino)butylic acid,2-amino-4-hydroxy-3-(2-amino-1-iminoethylimino)butylic acid,4-amino-2-hydroxy-3-(2-amino-1-iminoethylimino)butylic acid,2,4-dihydroxy-3-(2-amino-1-iminoethylimino)butylic acid,2,4-diamino-3-(2-hydroxy-1-iminoethylimino)butylic acid,

[0133] 2-amino-4-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylic acid,4-amino-2-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylic acid,2,4-dihydroxy-3-(2-hydroxy-1-iminoethylimino)butylic acid,2,4-diamino-3-(2-amino-1-iminoethylimino)butylamide,2-amino-4-hydroxy-3-(2-amino-1-iminoethylimino)butylamide,

[0134] 4-amino-2-hydroxy-3-(2-amino-1-iminoethylimino)butylamide,2,4-dihydroxy-3-(2-amino-1-iminoethylimino)butylamide,2,4-diamino-3-(2-hydroxy-1-iminoethylimino)butylamide,2-amino-4-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylamide,4-amino-2-hydroxy-3-(2-hydroxy-1-iminoethylimino)butylamide,2,4-dihydroxy-3-(2-hydroxy-1-iminoethylimino)butylamide and the like.

[0135] Examples of compounds represented by formula (3) above alsoinclude addition polymerization compounds in which the nitrile group ofglycinonitrile or glycolonitrile is addition-bonded to either the methylgroup or the imino group of the above-mentioned compounds. Suchcompounds represented by formula (3) above can have a random copolymerconfiguration wherein the structural unit represented by formula (4)above and the structural unit represented by formula (5) above arealternately arranged in the molecule, or a block copolymer configurationwherein the molecule contains at least two different polymer blocks,each of which independently contains at least one of the structuralunits represented by formulae (4) and (5).

[0136] As examples of addition cyclization compounds represented byformula (6) above which have a pyrimidine skeleton formed by thecyclization of a terminal having a nitrile group, there can be mentioned4,5-diamino-2,6-bis(aminomethyl)pyrimidine,4-amino-5-hydroxy-2,6-bis(aminomethyl)pyrimidine,5-amino-4-hydroxy-2,6-bis(aminomethyl)pyrimidine,4,5-diamino-2-aminomethyl-6-hydroxymethylpyrimidine,4-amino-5-hydroxy-2-aminomethyl-6-hydroxypyrimidine,5-amino-4-hydroxy-2-aminomethyl-6-hydroxypyrimidine,4,5-diamino-6-aminomethyl-2-hydroxymethylpyrimidine,4-amino-5-hydroxy-6-aminomethyl-2-hydroxypyrimidine,5-amino-4-hydroxy-6-aminomethyl-2-hydroxypyrimidine,4,5-diamino-2,6-bis(hydroxymethyl)pyrimidine,4-amino-5-hydroxy-2,6-bis(hydroxymethyl)pyrimidine,5-amino-4-hydroxy-2,6-bis(hydroxymethyl)pyrimidine and the like. Furtherexamples of compounds represented by formula (6) above include additioncyclization compounds formed by a reaction in which a nitrile group isaddition-bonded to a methylene group at the 1-position of any of theabove-mentioned compounds, and the resultant compound is substitutedwith the substituent represented by formula (8) or (9) above. Suchcompounds represented by formula (6) above can have a random copolymerconfiguration wherein the structural unit represented by formula (4)above and the structural unit represented by formula (5) above arealternately arranged in the molecule, or a block copolymer configurationwherein the molecule contains at least two different polymer blocks,each of which independently contains at least one of the structuralunits represented by formulae (4) and (5). In addition, the compoundsformed by the hydrolysis of the nitrile group of the above-mentionedaddition cyclization compounds can also be mentioned as examples ofcompounds represented by formula (6).

[0137] As examples of addition cyclization compounds represented byformula (7) above which have a pyridine skeleton formed by thecyclization of a terminal having a nitrile group, there can be mentioned2,3,4,5-tetra-amino-6-aminomethylpyridine,2,3,4-triamino-5-hydroxy-6-aminomethylpyridine,2,4,5-triamino-3-hydroxy-6-aminomethylpyridine,2,4,5-triamino-3,5-dihydroxy-6-aminomethylpyridine,2,3,4,5-tetramino-6-hydroxymethylpyridine,2,3,4-triamino-5-hydroxy-6-hydroxymethylpyridine,2,4,5-triamino-3-hydroxy-6-hydroxymethylpyridine and2,4,5-triamino-3,5-dihydroxy-6-hydroxymethylpyridine. Further examplesof compounds represented by formula (7) above include additioncyclization compounds formed by a reaction in which a nitrile group isaddition-bonded to a methylene group at the 6-position of any of theabove-mentioned compounds, and the resultant compound is substitutedwith the substituent represented by formula (8) or (9) above. Suchcompounds represented by formula (7) above can have a random copolymerconfiguration wherein the structural unit represented by formula (4)above and the structural unit represented by formula (5) above arealternately arranged in the molecule, or a block copolymer configurationwherein the molecule contains at least two different polymer blocks,each of which independently contains at least one of the structuralunits represented by formulae (4) and (5). In addition, the compoundsformed by the hydrolysis of the nitrile group of the above-mentionedaddition cyclization compounds can also be mentioned as examples ofcompounds represented by formula (7).

[0138] (C) At least one compound selected from the group consisting ofthe following compounds (c) and (d):

[0139] (c) a compound represented by the following formula (10) or (11):

[0140] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group, and

[0141] (d) a compound represented by the following formula (12):

(HCN)_(n)  (12)

[0142] wherein n represents an integer of 4 to 200.

[0143] Compounds represented by formula (10) or (11) above are atetramer of hydrogen cyanide and a compound formed by the hydrolysis ofthe nitrile group of the tetramer. Compounds represented by formula (12)above are polymers of hydrogen cyanide.

[0144] Examples of compounds represented by formula (10) or (11) above(i.e., tetramers of hydrogen cyanide) include diaminomaleonitrile and atautomer thereof, namely aminoiminosuccinonitrile. Examples of compoundsformed by the hydrolysis of the nitrile group of the tetramers ofhydrogen cyanide include diaminomaleic acid, 2,3-diamino-3-cyanoacrylicacid, 2,3-diamino-3-cyanoacrylamide, 2,3-diamino-3-carbamoylacrylicacid, 2,3-diamino-3-carbamoylacrylamide, aminoiminosuccinonic acid,2-amino-3-imino-3-carbamoylpropionamide,2-amino-3-imino-3-cyanopropionic acid,2-amino-3-imino-3-cyanopropionamide,2-amino-3-imino-3-carbamoylpropionic acid,

[0145] 3-amino-2-imino-3-cyanopropionic acid,3-amino-2-imino-3-cyanopropionamide and3-amino-2-imino-3-carbonylpropionic acid.

[0146] Examples of hydrogen cyanide polymers represented by formula (12)above include the following compounds. When n=4, in addition to thecompounds represented by formula (10) or (11) above, there can bementioned 3-amino-2,4-diiminobutylic acid; when n=5, there can bementioned 3-amino-3-cyano-2,4-diiminobutylic acid and3-amino-2,4,5-triiminohepthanoic acid; when n=6, there can be mentioned3,4-diamino-3,4-dicyano-2-iminobutylic acid,3-amino-3-cyano-2,4,5-triiminohepthanoic acid and the like. Furtherexamples of hydrogen cyanide polymers represented by formula (12)include hydrogen cyanide polymers in which n is 7 or more.

[0147] (D) A compound having in a molecule thereof at least one skeletonselected from the group consisting of the following skeletons (e) and(f):

[0148] (e) a skeleton represented by the following formula (13):

[0149] wherein each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; and n represents an integer of 2 to120,

[0150] and

[0151] (f) a skeleton represented by the following formula (14) or (15):

[0152] wherein:

[0153] each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group;

[0154] each Y² independently represents an amino group or a hydroxylgroup; and

[0155] n represents an integer of 1 to 70.

[0156] Examples of compounds having in a molecule thereof a skeletonrepresented by formula (13) above include a polymer of hydrogen cyanideand a polymer formed by the hydrolysis of the nitrile group of thehydrogen cyanide polymer. As a specific example of such a hydrogencyanide polymer, there can be mentioned a polymer which has at least onestructure selected from the group consisting of an aminocyanomethylenestructure, an aminocarbamoylmethylene structure and anaminocarboxymethylene structure and which has 2 or more recurring units.The hydrogen cyanide polymer mentioned above may be a compound whichexhibits a peak between 70 ppm and 90 ppm in a ¹³C-NMR spectrum asmeasured in heavy water, wherein the peak is ascribed to a methylenestructure.

[0157] Examples of compounds having in a molecule thereof a skeletonrepresented by formula (14) or (15) above include a polycyclic compoundand a compound formed by the hydrolysis of the nitrile group of thepolycyclic compound. As specific examples of such compounds, there canbe mentioned a polycyclic compound in which the 5-position and6-position of a 6-membered ring having 3,4,4,5-tetramino-4-(cyano,carboxy or carbamoyl)-3,4,5,6-tetrahydropyridine skeleton arerespectively the 2-position and 3-position of an adjacent 6-memberedring, and a polycyclic compound in which the 6-position, 7-position,8-position and 9-position of a3,7,9,10-tetramino-3,4,6,7,9,10-hexahydropyridino[2,3-e] pyridineskeleton are respectively the 5-position, 10-position, 4-position and3-position of an adjacent pyridine skeleton. Such a polycyclic compoundmay be a compound which exhibits a peak between 60 ppm and 80 ppm in a¹³C-NMR spectrum as measured in heavy water, wherein the peak isascribed to a methylene structure. Such a polycyclic compound may be acompound which exhibits absorption maximums at 380 nm and 460 nm in anultraviolet-visible absorption spectrum.

[0158] The mechanism of the production of the above-mentioned hydrogencyanide polymers and the like is described in “Kogyo Kagaku Zasshi(Journal of Industrial Chemistry)”, Volume 70, page 54 (1967) which ispublished by Japanese Chemical Society, Japan.

[0159] (E) A compound having in a molecule thereof at least one skeletonselected from the group consisting of the following skeletons (g) and(h):

[0160] (g) a skeleton represented by the following formula (16):

[0161] wherein:

[0162] each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group;

[0163] each Y² independently represents an amino group or a hydroxylgroup;

[0164] and n represents an integer of 2 to 120,

[0165] and

[0166] (h) a skeleton represented by the following formula (17) or (18):

[0167] wherein:

[0168] each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group;

[0169] each Y² independently represents an amino group or a hydroxylgroup; and

[0170] n represents an integer of 1 to 70.

[0171] The compounds having in a molecule thereof a skeleton representedby formula (16) above are a compound formed by oxidative polymerization(i.e., an oxidative polymerization product) and a compound formed by thehydrolysis of the nitrile group of the oxidative polymerization product.These compounds are known to be produced by an oxidative couplingreaction of methylene groups. Examples of such compounds include2,3-diaminosuccinonitrile and 2-amino-3-hydroxysuccinonitrile (which areformed by oxidative coupling of glycolonitrile and glycinonitrile); andcompounds formed by the hydrolysis of the above-mentioned compounds,such as 2,3-diaminosuccinonic acid, 2,3-diamino-3-carbamoylpropionamide,2,3-diamino-3-cyanopropionic acid, 2,3-diamino-3-carbamoylpropionicacid, 2,3-diamino-3-cyanopropionamide,2-amino-3-hydroxy-3-cyanopropionic acid,2-amino-3-hydroxy-3-carbamoylpropionic acid,2-amino-3-hydroxy-3-cyanopropionamide,3-amino-2-hydroxy-3-cyanopropionic acid,3-amino-2-hydroxy-3-carbamoylpropionic acid and3-amino-2-hydroxy-3-cyanopropionamide. Further examples of compounds (E)include oxidative polymerization products which have a structure whereina methylene compound is coupled to any of the above-mentioned compoundswhich have in a molecule thereof a skeleton represented by formula (16),and also include the compounds formed by the hydrolysis of the nitrilegroup of such oxidative polymerization products. A specific example ofsuch a compound is a compound which has at least one structure selectedfrom the group consisting of an aminocyanomethylene structure, anaminocarbamoylmethylene structure, an aminocarboxymethylene structure, ahydroxycyanomethylene structure, a hydroxycarbamoylmethylene structureand a hydroxyaminocarboxymethylene structure, and which has 2 or morerecurring units. Such an oxidative polymerization product mentionedabove may be a compound which exhibits a peak between 70 ppm and 90 ppmin a ¹³C-NMR spectrum as measured in heavy water, wherein the peak isascribed to a methylene structure.

[0172] A compound having in a molecule thereof a skeleton represented byformula (17) or (18) is formed by an addition cyclization of the cyanogroup of the above-mentioned oxidative polymerization product. Suchcompounds are a polycyclic compound having in a molecule thereof askeleton represented by formula (17) or (18) above and a compound formedby the hydrolysis of the nitrile group of the polycyclic compound. Asexamples of such compounds, there can be mentioned a polycyclic compoundin which the 5-position and 6-position of a 6-membered ring having a3,4,4,5-(amino and/or hydroxy)-4-(cyano, carboxy orcarbamoyl)-3,4,5,6-tetrahydropyridine skeleton are respectively the2-position and 3-position of an adjacent 6-membered ring, and apolycyclic compound in which the 6-position, 7-position, 8-position and9-position of a 3,7,9,10-(amino and/orhydroxy)-3,4,6,7,9,10-hexahydropyridino[2,3-e] pyridine skeleton arerespectively the 5-position, 10-position, 4-position and 3-position ofan adjacent pyridine skeleton. Such a polycyclic compound may be acompound which exhibits a peak between 60 ppm and 80 ppm in a ¹³C-NMRspectrum as measured in heavy water, wherein the peak is ascribed to amethylene structure. In addition, such a polycyclic compound may be acompound which exhibits absorption maximums between 340 nm and 380 nmand between 440 nm and 480 nm in an ultraviolet-visible absorptionspectrum.

[0173] (F) Hexamethylenetetramine and a compound having atrimethyleneamine structure which has a skeleton represented by thefollowing formula (1):

[0174] wherein n represents an integer of 1 to 150.

[0175] Such compounds are known to be produced by the condensation offormaldehyde and ammonia. Examples of compounds having a structurerepresented by formula (1) above include the following compounds. Whenn=1, there can be mentioned N-aminomethyl-hexahydro-1,3,5-triazine; whenn=2, there can be mentionedN,N′-bisaminomethyl-hexahydro-1,3,5-triazine, pentamethylenetetramineand hexamethylenepentamine; when n=3, there can be mentionedN,N′,N″-trisaminomethylhexahydro-1,3,5-triazine; and when n=4, there canbe mentioned hexamethylenetetramine and heptamethylene pentamine.

[0176] As explained above in detail, examples of organic impuritycompounds, which are contained in the hydrolysis reaction system used inthe present invention, include a compound formed by a condensationreaction between a hydroxyl group and an amino group, a compound formedby an addition reaction between a nitrile group and an activatedmethylene group, a compound formed by a cyclization reaction between anitrile group and an activated methylene group, a compound formed by anoxidation reaction of a methylene group, a compound formed by acyclization reaction of a methylene group, and a compound formed by thehydrolysis of a nitrile group of the above-mentioned compounds. Furtherexamples of organic impurity compounds include compounds formed by acombination of the above-mentioned reactions, namely the followingcompounds (I) to (V):

[0177] (I) a compound formed by a condensation reaction between an aminogroup of either a compound represented by formula (3), (6), (7), (10) or(11) or a skeleton represented by any one of formulae (13) to (18) and ahydroxyl group of either a compound represented by formula (3), (6) or(7) or a skeleton represented by any one of formulae (14) to (18);

[0178] (II) a compound formed by an addition reaction of a nitrile groupof either a compound represented by any one of formulae (2), (3) and(10) to (12) or a skeleton represented by formula (13), (14), (16) or(17);

[0179] (III) a compound formed by an addition reaction between a nitrilegroup of either a compound represented by any one of formulae (2), (3)and (10) to (12) or a skeleton represented by formula (13), (14), (16)or (17) and an imino group of a compound represented by formula (3),(6), (7) or (11);

[0180] (IV) a compound formed by an addition reaction between a nitrilegroup of either a compound represented by any one of formulae (2), (3)and (10) to (12) or a skeleton represented by formula (13), (14), (16)or (17) and a methylene group of a compound represented by any one offormulae (1) to (3), (6) and (7); and

[0181] (V) a compound formed by an oxidative coupling of a methylenegroup of one or more of the compounds represented by any one of formulae(1) to (3), (6) and (7).

[0182] Such a compound may be a compound which exhibits a peak between53 ppm and 100 ppm in a ¹³C-NMR spectrum as measured in heavy water.Further, since a compound produced by a cyclization reaction isdiscolored, a cyclization compound as an organic impurity compound maybe a compound which exhibits absorption maximums between 340 nm and 380nm and between 440 nm and 480 nm in an ultraviolet-visible absorptionspectrum.

[0183] The organic impurity compound contained in the hydrolysisreaction system used in the present invention is a polyfunctionalcompound having a molecular weight of 95 or more and such a compoundmarkedly lowers the activity of a microbial enzyme. In the presentinvention, glycine is produced under conditions wherein the content ofthe organic impurity compound having a molecular weight of 95 or more,preferably 130 or more, in the hydrolysis reaction system is maintainedat a level of 10% by weight or less, preferably 5% by weight or less,more preferably 1% by weight or less, based on the weight of thehydrolysis reaction system, so as to improve the activity of themicrobial enzyme.

[0184] With respect to the method for maintaining the content of theorganic impurity compound in the hydrolysis reaction system at a levelof 10% by weight or less, based on the weight of the hydrolysis reactionsystem, there can be mentioned a method wherein the amount of oxygendissolved in the hydrolysis reaction system is suppressed to 5 ppm byweight or less, based on the weight of the hydrolysis reaction system.The amount of oxygen dissolved in the hydrolysis reaction system underatmospheric pressure is generally 8 ppm to 10 ppm by weight. When theamount of oxygen dissolved in the hydrolysis reaction system issuppressed to 5 ppm by weight or less, the formation of the by-productsis lowered. For suppressing the inhibition of the hydrolytic activity ofthe microbial enzyme and the like, it is preferred that the amount ofoxygen dissolved in the hydrolysis reaction system is 0.5 ppm by weightor less, more advantageously 0.05 ppm by weight or less, based on theweight of the hydrolysis reaction system.

[0185] In the present invention, as a method for suppressing the amountof oxygen dissolved in the hydrolysis reaction system, a method can beemployed in which the hydrolysis is conducted using a closed reactionsystem which is sealed off from the air, a reaction system which ispressurized with an inert gas, a reaction system through which an inertgas is flowed, or a reaction system which is under a pressure of lessthan atmospheric pressure. As examples of inert gases, there can bementioned noble gases, such as helium gas and argon gas; natural gases,such as methane gas, ethane gas and propane gas; low boiling pointethers, such as diethyl ether; and nitrogen gas. These gasses can beused individually or in combination. From the viewpoint of safety andthe like, nitrogen gas is preferred. In the case wherein a closedreaction system is used, sealing off of the reaction system from the aircan be more effectively performed by pressurizing the reaction systemwith an inert gas. With respect to the pressure level to which thereaction system should be pressurized, there is no particularlimitation. However, in view of the pressure resistance of the microbialenzyme used, it is preferred that the pressure of the pressurizedreaction system is in the range of from superatmospheric pressure to 1.0MPa, more advantageously superatmospheric pressure to 0.5 MPa. On theother hand, when a reaction system which is under a pressure of lessthan atmospheric pressure is used, the pressure of the reaction systemcan be in the range of from more than 0.7 kPa (0.7 kPa is the vaporpressure of water at 0° C.) to less than atmospheric pressure,preferably from more than 4.0 kPa to less than atmospheric pressure.

[0186] In the present invention, for suppressing the amount of oxygendissolved in the hydrolysis reaction system, it is necessary to not onlyseal off the reaction system from the air, but also remove oxygen whichis dissolved in the raw materials and the like. Various conventionalmethods can be employed for removing oxygen from the reaction system.For example, there can be mentioned a distillation deaeration method inwhich oxygen dissolved in the reaction system is removed bydistillation; an inert gas flow treatment method in which an inert gasis flowed through the reaction system to thereby replace oxygendissolved in the system by an inert gas; and a reductive compoundaddition treatment method in which a reductive compound is added to thereaction system to thereby consume oxygen by reduction. Examples ofreductive compounds which can be used for the reductive compoundaddition treatment include reductive biochemical compounds, compounds offormic acid and compounds of sulfurous acid. With respect to thereductive biochemical compounds which can be used for the reductivecompound addition treatment, there is no particular limitation. Forexample, there can be used L-ascorbic acid; L-ascorbic acid esters, suchas L-ascorbic acid stearic acid ester; salts, such as sodiumL-ascorbate; glutathione; L-cysteine; cysteine salts, such as L-cysteinehydrochloride monohydrate; cysteine esters, such as L-cysteine ethylester chloride and L-cysteine methyl ester chloride; and N-substitutedcysteines, such as N-acetyl-L-cysteine. Among these compounds,L-ascorbic acid is preferred. The amount of the reductive biochemicalcompound added to the reaction system varies depending on the amount ofoxygen dissolved in the system; however, from the viewpoint of thestabilization of glycinonitrile, the reductive biochemical compound isadded in an amount of 0.001 to 5 mol %, preferably 0.01 to 2 mol %,based on the molar amount of glycinonitrile. With respect to thereductive compounds of formic acid which can be used for the reductivecompound addition treatment, there is no particular limitation. Forexample, there can be used formic acid; formic acid salts, such asammonium formate; and formic acid esters, such as methyl formate andethyl formate. Among these compounds, formic acid and ammonium formateare preferred. The amount of the reductive compound of formic acid addedto the reaction system varies depending on the amount of oxygendissolved in the system; however, from the viewpoint of thestabilization of glycinonitrile, the reductive compound of formic acidis added in an amount of 0.002 to 8 mol %, preferably 0.02 to 4 mol %,based on the molar amount of glycinonitrile. With respect to thecompounds of sulfurous acid which can be used for the reductive compoundaddition treatment, there is no particular limitation. For example,there can be used sulfur dioxide, sulfurous acid, sodium sulfite, sodiumhydrogen sulfite, potassium sulfite, potassium hydrogen sulfite andammonium sulfite. Among these compounds, ammonium sulfite is preferred.Since the compounds of sulfurous acid are electrolytes and exhibit theeffect to promote the production of by-products, the compound ofsulfurous acid is added to the reaction system in an amount of 0.001 to2% by weight, preferably 0.01 to 1% by weight, wherein the amount shouldbe selected so as to meet the below-described requirement of the totalamount of the electrolytes in the reaction system.

[0187] Further, as another method for maintaining the content of theorganic impurity compound in the hydrolysis reaction system at a levelof 10% by weight or less, based on the weight of the hydrolysis reactionsystem, there can be mentioned a method wherein ammonia is used as astabilizer for glycinonitrile. When glycinonitrile is heated, ammoniaand imine compounds (such as iminodiacetonitrile) are generated andfurther heating causes the imine compounds to denature, resulting in thegeneration of black compounds. Therefore, glycinonitrile can bestabilized by using a hydrolysis reaction system having ammoniadissolved therein. The amount of ammonia to be added to the reactionsystem may vary depending on the reaction temperature and reaction time,but it is preferred that ammonia is used in an amount of 0.001 to 5 mol,more advantageously 0.01 to 2 mol, per mol of glycinonitrile per liter.In general, excess ammonia remains in glycinonitrile after the synthesisthereof. The excess ammonia as such can be used as a stabilizer withoutseparating the excess ammonia from glycinonitrile.

[0188] In addition, as a further method for maintaining the content ofthe organic impurity compound in the hydrolysis reaction system at alevel of 10% by weight or less, based on the weight of the hydrolysisreaction system, there can be mentioned a method wherein the amount ofan electrolyte in the reaction system is maintained at a level of 2% byweight or less, based on the weight of the glycinonitrile. Electrolytesare known to promote the production of by-products from nitrilecompounds. Examples of electrolytes used in the present inventioninclude not only alkali metal salts, alkaline earth metal salts andmixtures of two or more metal salts (such as phosphate buffers), butalso alkali metal compounds and alkaline earth metal compounds (such ascaustic soda) which are capable of forming glycine salts, and mineralacids (such as sulfuric acid, hydrochloric acid and nitric acid). Theaddition of such electrolytes to the reaction system promotes theformation of by-products from nitrile compounds, and, as one of thereasons for such promotion, it is noted that the addition of anelectrolyte to an aqueous solution causes an increase in the amount ofoxygen dissolved in the aqueous solution (see “Kagaku Benran (ChemistryHandbook)”, revised 4th edition, page II-159, Table 8.54, published byJapanese Chemical Society, Japan). Therefore, it is preferred thatbefore using the microbial enzyme, an electrolyte which is used as aculture medium or a buffer in the production of the microbial enzyme andwhich remains in the microbial enzyme is reduced to a predeterminedlevel by washing, that the amount of an alkali catalyst contained in anaqueous glycinonitrile solution is decreased, and that the amount of aphosphate buffer added to a reaction medium liquid for adjusting the pHvalue thereof is suppressed to a level as low as possible. The totalamount of the electrolytes contained in the hydrolysis reaction systemis adjusted to 2% by weight or less, preferably 1% by weight or less,more preferably 0.5% by weight or less, based on the weight of theglycinonitrile.

[0189] The above-mentioned methods in which the amount of oxygendissolved in the hydrolysis reaction system is suppressed, theabove-mentioned method in which ammonia is dissolved into the hydrolysisreaction system, and the above-mentioned method in which the electrolyteconcentration of the hydrolysis reaction system is controlled can beused in combination. It is preferred that all these methods are used incombination.

[0190] With respect to the microorganism which is used to produce themicrobial enzyme used in the present invention wherein the enzyme hasthe activity to hydrolyze a nitrile group, there is no particularlimitation. For example, there can be suitably used a microorganismbelonging to a genus selected from the group consisting ofAcinetobacter, Rhodococcus, Corynebacterium, Alcaligenes, Mycobacterium,Rhodopseudomonas and Candida. However, the microorganism is not limitedto them. Specific examples of microorganisms include the followingmicrobial strains (1) to (9):

[0191] (1) Acinetobacter sp. AK226 (see Examined Japanese PatentApplication Publication Specification No. Sho 63-2596). deposited withthe National Institute of Bioscience and Human-Technology, Japan (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Postal Code No.305-0046)) on May 28, 1985 (original deposit date) under the accessionnumber FERM BP-2451;

[0192] (2) Acinetobacter sp. AK227 (see Examined Japanese PatentApplication Publication Specification No. Sho 63-2596), deposited withthe National Institute of Bioscience and Human-Technology, Japan (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Postal Code No.305-0046)) on May 28, 1985 (original deposit date) under the accessionnumber FERM BP-7413;

[0193] (3) Rhodococcus maris BP-479-9 (see Unexamined Japanese PatentApplication Laid-Open Specification Nos. Hei 7-303491 and Hei 7-303496),deposited with the National Institute of Bioscience andHuman-Technology, Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,Japan (Postal Code No. 305-0046)) on Nov. 2, 1993 (original depositdate) under the accession number FERM BP-5219;

[0194] (4) Corynebacterium sp. C5 (see Examined Japanese PatentApplication Publication Specification No. Hei 6-65313), deposited withthe National Institute of Bioscience and Human-Technology, Japan (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Postal CodeNo.305-0046)) on Aug. 28, 1986 (original deposit date) under theaccession number FERM BP-7414;

[0195] (5) Corynebacterium nitrilophilus (see Unexamined Japanese PatentApplication Laid-Open Specification No. Hei 2-84198), deposited with theAmerican Type Culture Collection, U.S.A. (University Boulevard, ManassasVa. (Postal Code No. 10801)) under the accession number ATCC 21419;

[0196] (6) Alcaligenes faecalis IFO 13111 (see Unexamined JapanesePatent Application Laid-Open Specification No. Hei 6-70856), depositedwith the National Institute of Bioscience and Human-Technology, Japan(1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Postal Code No.305-0046)) on Jul. 22, 1994 (original deposit date) under the accessionnumber FERM BP-4750;

[0197] (7) Mycobacterium sp. AC777 (see Unexamined Japanese PatentApplication Laid-Open Specification No. Hei 2-84918 and U.S. Pat. No.5,283,193), deposited with the National Institute of Bioscience andHuman-Technology, Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,Japan (Postal Code No.305-0046)) on Mar. 27, 1989 (original depositdate) under the accession number FERM BP-2352;

[0198] (8) Rhodopseudomonas spheroides (see Unexamined Japanese PatentApplication Laid-Open Specification No. Hei 6-303991), deposited withthe American Type Culture Collection, U.S.A. (University Boulevard,Manassas Va. (Postal Code No. 10801)) under the accession number ATCC11167; and

[0199] (9) Candida tropicalis (see Unexamined Japanese PatentApplication Laid-Open Specification No. Hei 6-303991), deposited withthe American Type Culture Collection, U.S.A. (University Boulevard,Manassas Va. (Postal Code No. 10801)) under the accession number ATCC20311.

[0200] The conventional culture medium can be used for culturing amicroorganism which produces the microbial enzyme used in the presentinvention. As a carbon source, there can be used, for example, glucose,glycerin, saccharose, fractose, organic acids (such as acetic acid), adextrin and maltose. As a nitrogen source, there can be used ammonia andsalts thereof, urea, sulfuric acid salts, nitric acid salts, organicnitrogen sources (such as yeast extract, malt extract, peptone, soy oiland meat extract). In addition, inorganic nutrients, such as phosphoricacid salts, sodium, potassium, iron, magnesium, cobalt, manganese andzinc, and vitamins are appropriately selected and added to the culturemedium. The microorganism is cultured aerobically at a pH value of 4 to10, preferably 6 to 8, at 5 to 50° C., preferably 20 to 35° C. Further,enzyme inducers, such as lactam compounds (γ-lactam, δ-lactam,ε-caprolactam and the like), nitrile compounds and amide compounds, maybe added to the culture medium. With respect to the microorganism usedfor producing the microbial enzyme used in the present invention in thecase of the commercial practice of the present invention, themicroorganism in original form can be used for producing the microbialenzyme, or a mutant strain derived therefrom can be used for producingthe microbial enzyme. For example, a mutant strain which produces theenzyme constitutively can be obtained by inducing a spontaneous mutationusing an appropriate mutagen or by genetic engineering techniques. Asthe microbial enzyme used in the present invention, there can be usedeither of the following products: the cells of a microorganism(microbial cells) which have been recovered from a culture broth, and amicrobial preparation. Examples of microbial preparations include ahomogenate of a microorganism, an enzyme separated from the homogenate,an immobilized microorganism, and an immobilized enzyme which isobtained by separating and extracting an enzyme from a microorganism andimmobilizing the extracted enzyme on a carrier. The microbial cells canbe recovered from the culture broth by a conventional method.

[0201] In the present invention, the microbial cells or microbialpreparation obtained in the above-mentioned manner can be stored in theform of an aqueous microbial suspension which is prepared by suspendingthe microbial cells or microbial preparation in distilled water or abuffer. For decreasing the amount of wastes discarded after thereaction, it is preferred that the above-mentioned microbial suspensionis prepared using distilled water, especially recycled distilled waterwhich is obtained by a method in which, during the crystal-depositionoperation and the like, distillation is performed under conditionswherein the reaction mixture is completely sealed off from the air. Forimproving the storage stability, a stabilizer may be added to themicrobial suspension. For decreasing the amount of wastes discardedafter the reaction, glycine is preferably used as the above-mentionedstabilizer.

[0202] In the present invention, by using any one of the below-mentionedmethods, the hydrolysis of glycinonitrile to produce glycine can becaused to proceed quickly: a method in which the aqueous glycinonitrilesolution or the ammonia-containing aqueous glycinonitrile solution(obtained in the above-mentioned manner) is added to the aqueoussuspension of microbial cells or microbial preparation (obtained in theabove-mentioned manner); a method in which the aqueous suspension ofmicrobial cells or microbial preparation is added to the aqueousglycinonitrile solution; and a method in which the microbial cells ormicrobial preparation (obtained in the above-mentioned manner) isimmobilized on a carrier, and the aqueous glycinonitrile solution iscaused to flow while contacting the immobilized microbial cells ormicrobial preparation. In general, 0.01 to 5% by weight of theabove-mentioned microbial cells or microbial preparation, in terms ofthe dry weight of the microorganism, and 1 to 40% by weight ofglycinonitrile (enzyme substrate) are charged into a reactor and areaction is performed at a temperature of, for example, 0 to 60° C.,preferably 10 to 50° C., for 1 to 24 hours, preferably 3 to 8 hours.There may be used a reaction method wherein glycinonitrile is chargedinto the reactor to obtain a low concentration of glycinonitrile, andadditional glycinonitrile is then gradually charged in the course of thereaction, or a reaction method wherein the reaction temperature ischanged with time. When glycinonitrile is hydrolyzed into glycine andammonia, the pH value of the reaction system becomes high, as comparedto the pH value of the reaction system before initiating the hydrolysisreaction. For preventing the pH value of the reaction system fromincreasing in accordance with the progress of the reaction, a buffer maybe added to the reaction system before initiating the reaction, oralternatively, an acid or alkali may be added to the reaction systemduring the reaction. However, from the viewpoint of decreasing theamount of wastes, it is preferred that a buffer, acid or alkali is notadded to the hydrolysis reaction system. The hydrolysis can be conductedin an open type reactor; however, from the viewpoint of preventing theproblem that the by-produced ammonia is dispersed into the air to causeenvironmental pollution, viewpoint of efficient recovering of ammonia,and viewpoint of efficient sealing off of the reaction system from theair, it is preferred that the hydrolysis reaction is conducted using aclosed reaction system in a sealed reactor so as to cause theby-produced ammonia to be accumulated in the reactor.

[0203] The ammonia and glycine can be separately recovered by at leastone operation selected from the group consisting of distillation,reactive distillation, entrainment by inert gas, ion exchange,extraction, reprecipitation using a poor solvent, and crystal-depositionby concentration or cooling. From the viewpoint of ease in the operationand of avoiding the use of an organic solvent, acid and alkali, it ispreferred that first, ammonia is recovered by distillation, reactivedistillation or entrainment by an inert gas, and then, a liquidremaining after the recovery of the ammonia is subjected tocrystal-deposition by concentration or cooling to thereby recoverglycine. As a reactor for conducting a reactive distillation, there canbe used a single column tower, a multi-stage tower or a packed towerequipped with a cooler for recovering ammonia and water by cooling. Itis preferred that the reactive distillation is conducted in a continuousmanner or intermittent manner under a pressure which is equal to orhigher than the boiling pressure of the reaction mixture, for example,20.0 kPa or higher at 60° C., and 0.6 kPa or higher at 0° C. It is morepreferred that vacuum reactive distillation is conducted under apressure of 12.6 kPa to 1.3 kPa. When ammonia is recovered byentrainment by an inert gas, the recovery of ammonia from the reactionmixture can be performed by a method in which a reactor is used which isequipped with a blow nozzle for introducing an inert gas and a coolingtrap for recovering ammonia and water from the inert gas, and ammonia iscaused to be entrained by an inert gas in a continuous manner orintermittent manner under a pressure which is in the range of from aslightly superatmospheric pressure to a reduced pressure, therebyseparating ammonia from the reaction mixture. Further, for promoting theseparation of ammonia, vacuum reactive distillation can be performed ina flow of an inert gas. The reaction mode can be a batchwise mode or aflow reaction mode, or a combination of the two modes.

[0204] Thus, the reaction mode for the hydrolysis reaction may varydepending on the method for adding the microorganism or glycinonitrile,and the method for separating and recovering ammonia. When an aqueoussuspension of microbial cells or microbial preparation is used or whenammonia is recovered by reactive distillation or entrainment by an inertgas, in general, the hydrolysis reaction can be conducted mainly by abatchwise reaction mode or a multi-stage flow reaction mode whichemploys a multi-stage agitation vessel. When an immobilizedmicroorganism is used, the hydrolysis reaction can be conducted mainlyby a flow reaction mode which employs a tubular reactor. Theabove-mentioned reaction modes may be used in combination. The reactionmode for the hydrolysis reaction is not limited to those mentionedabove.

[0205] By the method of the present invention, glycinonitrile ishydrolyzed to obtain glycine in a yield of approximately 100 mol %. Allof the by-produced ammonia can be accumulated in a sealed reactor in theform of an ammonium salt of glycine, wherein the glycine in the reactoris present in the form of a highly concentrated aqueous glycinesolution. Alternatively, all or a most part of the by-produced ammoniacan be separated from the reaction mixture simultaneously with thereaction by reactive distillation or entrainment by an inert gas and theseparated ammonia can be recovered by cooling. When the microorganismcontains nitrile hydratase as a microbial enzyme, glycine is hydrolyzedby nitrile hydratase to produce glycine amide in the reaction system. Inthis case, by adding to the reaction system a microorganism or microbialenzyme having the activity to hydrolyze glycine amide, the glycine amidecan be completely converted into glycine and ammonia. Recovery ofglycine from the highly concentrated aqueous glycine solution containingammonium salts of glycine can be conducted, for example, by a methodcomprising separating the microorganism or microbial enzyme bycentrifugal filtration and/or membrane filtration, and recoveringglycine by crystal-deposition, ion exchange or reprecipitation using apoor solvent. The by-produced ammonia can be separated by evaporationtogether with a part of water and then recovered by distillation orextraction. A preferred embodiment of the method of the presentinvention for producing glycine comprises, for example, the followingsteps:

[0206] (1) reacting hydrogen cyanide with formaldehyde in an aqueousmedium in the presence of an alkali catalyst in a closed reaction systemto obtain glycolonitrile in the form of an aqueous solution thereof,

[0207] (2) adding ammonia to the aqueous solution of glycolonitrile toeffect a reaction between the glycolonitrile and the ammonia, to therebyobtain glycinonitrile in the form of an aqueous solution thereof whileproducing water,

[0208] (3) separating most of the ammonia and a part of the water fromthe obtained aqueous solution of glycinonitrile by distillation tothereby obtain a hydrolysis reaction system containing theglycinonitrile in the form of an aqueous solution thereof and theammonia remaining unseparated, wherein the separated ammonia isrecovered for recycle thereof to step (2),

[0209] (4) subjecting the hydrolysis reaction system to a hydrolysisreaction under the action of a microbial enzyme produced by amicroorganism added in the hydrolysis reaction system which is in aclosed system, thereby converting the glycinonitrile to glycine whileby-producing ammonia,

[0210] (5) separating the microorganism and the microbial enzyme by atleast one operation selected from the group consisting of centrifugalfiltration and membrane filtration, wherein the microorganism and themicrobial enzyme are recovered for recycle thereof to step (4),

[0211] (6) separating a part of organic impurity compounds inhibitingthe microbial enzyme which compounds are by-produced in steps (1) to (5)by at least one operation selected from the group consisting of membranefiltration and adsorbent-separation,

[0212] (7) separating by distillation the ammonia by-produced in step(4) and an excess amount of water which remains in the hydrolysisreaction system after step (4), wherein the separated ammonia isrecovered for recycle thereof to step (2),

[0213] (8) after or simultaneously with step (7), separating the glycineby crystal-deposition, and

[0214] (9) drying the crystals of the glycine.

[0215] In another aspect of the present invention, there is provided amethod for producing glycine, which comprises providing glycinonitrilein the form of an aqueous solution thereof, subjecting the aqueoussolution of glycinonitrile to a hydrolysis reaction, thereby convertingthe glycinonitrile to glycine while by-producing ammonia, and isolatingthe glycine from the hydrolysis reaction system, wherein the hydrolysisof glycinonitrile is conducted in the presence of ammonia. In thismethod, it is preferred that the amount of the ammonia is from 0.001 to5 mol, relative to 1 mole of the glycinonitrile. By the use of themethod of the present invention, it becomes possible to stabilizeglycinonitrile and produce glycine efficiently and stoichiometrically.

[0216] In a further aspect of the present invention, there is provided amethod for producing glycine, which comprises subjecting an aqueoussolution of glycinonitrile to a hydrolysis reaction, thereby convertingthe glycinonitrile to glycine while by-producing ammonia, and isolatingthe glycine from the hydrolysis reaction system, wherein the isolationof the glycine from the hydrolysis reaction system is conducted whilerecovering the by-produced ammonia separately from the recovery ofglycine in the absence of a base and an acid. In this case, it ispreferred that the glycine and ammonia are separately recovered by atleast one operation selected from the group consisting of distillation,reactive distillation, entrainment by an inert gas, ion exchange,extraction, reprecipitation using a poor solvent, and crystal-depositionby concentration or cooling. In addition, it is preferred that theammonia is recovered by distillation, reactive distillation orentrainment by an inert gas, and the glycine is recovered by subjectinga liquid remaining after the recovery of the ammonia tocrystal-deposition by concentration or cooling. By the use of the methodof the present invention, it becomes possible to recycle ammonia anddecrease the burden on the environment.

BEST MODE FOR CARRYING OUT THE INVENTION

[0217] Hereinbelow, the present invention will be described in moredetail with reference to the following Examples and Comparative Example,but they should not be construed as limiting the scope of the presentinvention.

[0218] In the Examples and Comparative Example, in order to prevent theentry of oxygen into a reaction system, all of the basic reactionoperations were conducted under a nitrogen atmosphere. In addition,before adding an aqueous solution to the reaction system for the firsttime, oxygen dissolved in the aqueous solution was purged with nitrogen(i.e., the oxygen concentration was decreased to 0.01 ppm or less) bysubjecting the aqueous solution to a nitrogen purge operation whichcomprises, for example, a several-time repetition of a cycle comprisingpressurizing the aqueous solution with nitrogen gas and then returningthe pressure to atmospheric pressure. However, it should be noted thatin a commercial scale process (Example 12), water used for preparingaqueous solutions is distilled water which has been recycled from a stepof concentrating glycinonitrile and from a step of concentration for thecrystal-deposition of glycine, and therefore, there is no need toconduct any special operations (for removing oxygen) other than storingthe aqueous solutions and the like in a nitrogen atmosphere.

[0219] In the following Examples and Comparative Example, variousoperations were conducted in the following manners.

[0220] (1) Evaporation and Recovery of Ammonia and Water

[0221] Evaporation and recovery of ammonia and water were conductedusing a thin film distillation apparatus (manufactured and sold byShibata Scientific Technology Inc., Japan) under a reduced pressure andat a residence time of 1 minute or less.

[0222] (2) Concentration of a Reaction Mixture

[0223] Concentration of 30 ml or less of a reaction mixture wasconducted using a rotary evaporator equipped with a circulatingaspirator for operating the rotary evaporator under reduced pressure(manufactured and sold by Tokyo Rikakikai Co., Ltd., Japan).

[0224] (3) Separation of a Microorganism

[0225] Separation of a microorganism after a hydrolysis reaction ofglycinonitrile was conducted by centrifuging a reaction mixture at10,000 rpm for 15 minutes using a refrigerating centrifuge (manufacturedand sold by Hitachi Ltd., Japan).

[0226] (4) Removal of Proteins

[0227] In Examples 1 to 11, proteins were removed by filtration underpressure using an ultrafiltration filter for syringes (manufactured andsold by Terumo Corp., Japan). In Example 12, proteins were removed usinga circulating ultrafiltration apparatus (Ultrafiltration membrane:“SIP-1013”, manufactured and sold by Asahi Kasei Kabushiki Kaisha,Japan).

[0228] (5) Liquid Chromatography

[0229] Glycine, glycinonitrile, glycolonitrile, iminodiacetonitrile,iminodiacetic acid, hexamethylenetetramine, ammonia, sulfuric acid ions,sodium ions and the like were analyzed by ion pair chromatography. As ananalytical instrument, use was made of a liquid chromatography apparatus(model LC-6A, an R1 and a UV detector, each manufactured and sold byShimadzu Corporation, Japan) and a column (ODS column, manufactured andsold by Tosoh Corp., Japan). In the ion pair chromatography, sodiumpentanesulfonate was used as an ion pair reagent.

[0230] (6) Gel Permeation Chromatography

[0231] Glycolonitrile having a molecular weight of 55 and organicimpurity compounds having a molecular weights of 4,000 or less wereanalyzed by gel permeation chromatography (GPC). As a gel permeationchromatography apparatus, use was made of model LC-9A (manufactured andsold by Shimadzu Corporation, Japan) and a GPC-IR, an RI and a UVdetector (each manufactured and sold by Nicolet Instrument Corporation,U.S.A.). As a column, use was made of “Asahipak GS-220 NQ column” (aShowdex column manufactured and sold by Showa Denko K.K., Japan).

[0232] (7) Analysis of a Discolored Compound

[0233] Using an ultraviolet-visible spectrophotometer (model U-3120,manufactured and sold by Hitachi, Ltd., Japan), a hydrolysis reactionsystem was subjected to the measurement of the ultraviolet-visibleabsorption spectrum between 200 nm and 800 nm. A discolored compoundcontained in the hydrolysis reaction system was analyzed by determiningthe wave length at an absorption maximum and the height of the peak.

[0234] (8) NMR

[0235] Organic impurity compounds were analyzed by measuring each of¹H-NMR spectrum and ¹³ C-NMR spectrum in heavy water. The ¹H-NMRanalysis was conducted using “JNM-α400” (manufactured and sold by JEOLLTD., Japan) and the ¹³C-NMR analysis was conducted using “AC-200”(manufactured and sold by Bruker Instruments, Germany). A sample for NMRwas prepared by drying the hydrolysis reaction system under reducedpressure to obtain a dried matter, dissolving the obtained dried matterin heavy water, and adding thereto 4,4-dimethyl-4-silane-pentanesulfonicacid sodium salt (δ=0.015 ppm) as an internal standard.

EXAMPLE 1

[0236] (1) Synthesis of Glycinonitrile

[0237] An 8-liter autoclave equipped with an agitator and a jacket(wherein the autoclave is manufactured and sold by Asahi Kasei KabushikiKaisha, Japan) was used as a reactor. The inside of the reactor waspurged with nitrogen gas, and 1,200 g of a 37% aqueous formaldehydesolution and hydrogen cyanide in an amount equivalent to the amount ofthe formaldehyde were added to the reactor, and the formaldehyde andhydrogen cyanide were reacted with each other to thereby obtain anaqueous glycolonitrile solution. To the obtained aqueous glycolonitrilesolution was added 5,000 g of a 25% aqueous ammonia solution, and areaction was performed for 2 hours, thereby obtaining a reaction mixturecontaining glycinonitrile synthesized. Next, unreacted ammonia andexcess water both present in the reaction mixture were removed underreduced pressure by means of a thin film distillation apparatus, tothereby obtain a 30% by weight aqueous glycinonitrile solution. Theobtained aqueous glycinonitrile solution was analyzed by liquidchromatography, and the analysis showed that the glycinonitrile solutioncontained 0.8% by weight of iminodiacetonitrile in addition toglycinonitrile. Further, the aqueous glycinonitrile solution wasanalyzed by gel permeation chromatography, and the analysis showed that1.2% by weight of at least one organic impurity compound having amolecular weight of 95 or more was contained in the aqueousglycinonitrile solution.

[0238] In addition, the aqueous glycinonitrile solution was analyzed by¹³C-NMR. It was found that in addition to the peaks ascribed toglycinonitrile and iminodiacetonitrile, a peak was present around 60 ppmto 70 ppm in the NMR spectrum. When the absorbance of the aqueousglycinonitrile solution was determined, it was found that absorptionmaximums were observed at 344 nm and 468 nm, and the absorbances at 344nm and 468 nm, in terms of a value as measured using a 10 mm quartzcell, were 0.297 and 0.08, respectively.

[0239] (2) Culturing of a Microorganism

[0240] Acinetobacter sp. AK226 (FERM BP-2451) was cultured at 30° C. for1 day in a medium having the following composition.

[0241] Medium

[0242] (The medium was prepared by dissolving the below-mentionedcomponents in distilled water, and had a pH value of 7.5) fumaric acid1.0% by weight meat extract 1.0 peptone 1.0 sodium chloride 0.1ε-caprolactam 0.3 potassium(I) phosphate 0.2 magnesium sulfateheptahydrate 0.02 ammonium chloride 0.1 iron(II) sulfate heptahydrate0.003 manganese chloride tetrahydrate 0.002 cobalt chloride hexahydrate0.002

[0243] (3) Hydrolysis of Glycinonitrile

[0244] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 18.0% by weight in terms of the dry weight of themicroorganism. The inside of a 200 ml glass autoclave was purged withnitrogen gas, and 53.3 g of the 30% by weight aqueous glycinonitrilesolution synthesized in step (1) above and 46.7 g of distilled waterwere added thereto under a nitrogen atmosphere. Subsequently, 1.0 g ofthe microbial suspension was added to the autoclave, and a reaction wasinitiated at 40° C. At this time, the pH value of the reaction system inthe autoclave was approximately 7. After the start of the reaction, thepH value of the reaction system began to increase. Two hours after thestart of the reaction, the pH value became 10. The reaction wasperformed for 10 hours, thereby obtaining a reaction mixture in anamount of 100 g. 2 g of the reaction mixture was taken out. With respectto the taken-out reaction mixture, the amounts of glycinonitrile as araw material and glycine produced were determined by liquidchromatography, and the amount of ammonia was determined by theNessler's method. The results of the liquid chromatography showed thatglycinonitrile used as a raw material had completely disappeared fromthe reaction system and the yield of glycine was 99%. Ammonia wasstoichiometrically produced. The amount of glycine produced per gram(dry weight) of the microorganism was 117 g/g and the activity forglycine production was 12 g/g·Hr.

[0245] The remaining 98 g of the reaction mixture was centrifuged whilecooling, to thereby separate the microorganism and recover asupernatant. The recovered supernatant was subjected to filtration underpressure by means of an ultrafiltration membrane to thereby remove theresidual cells of the microorganism and proteins from the supernatantand obtain a filtrate. 2 ml of the filtrate was taken out and subjectedto the measurement of the ultraviolet-visible absorption spectrum, andit was found that the absorbance at 468 nm was 0.79. The remainder ofthe filtrate was subjected to distillation by means of a thin filmdistillation apparatus to thereby separate ammonia and excess water fromthe filtrate by evaporation. As a result, when the condensate and theice and liquid recovered in a dry ice-ethanol trap were combinedtogether, 48 g of an aqueous ammonia solution was obtained. 43 g of aconcentrated glycine solution was recovered from the bottom of thedistillation apparatus. The concentrated glycine solution was cooled toroom temperature to thereby separate glycine by crystal-deposition. Thecrystals obtained by conducting the crystal-deposition operation onlyonce were dried, thereby obtaining 16.5 g of glycine crystals. Thepurity of the obtained glycine was 99.99%.

COMPARATIVE EXAMPLE 1

[0246] The influence of organic impurity compounds which are by-productscontained in an aqueous glycinonitrile solution was analyzed in thefollowing manner. A 30% by weight aqueous glycinonitrile solutionobtained in the same manner as in Example 1 was heated at 90° C. under aflow of nitrogen gas. The steam generated by heating and entrained bythe nitrogen gas was captured by a dry ice-ethanol trap. 10 ml of theheated glycinonitrile solution was sampled at the time points of 15minutes, 30 minutes and 1 hour after the start of the heating, and,thereafter, the sampling was further performed every 30 minutes until 5hours passed from the start of the heating. The solution samples (11samples in total) were stored under cooling.

[0247] A part of each of the solution samples was individually analyzedby liquid chromatography, gel permeation chromatography,ultraviolet-visible absorption spectrometry and ¹³C-NMR. Theglycinonitrile solution which was heated for 30 minutes or longer had anodor of ammonia. Although the glycinonitrile solution was concentratedin accordance with the lapse of the heating time, the heatedglycinonitrile solution had a glycinonitrile concentration within therange of from 30 to 35% by weight. From these results, it was found thatthe glycinonitrile contained in the glycinonitrile solution had beendenatured by the heating. Further, the amounts of organic impuritycompounds having molecular weights of 95 or more increased with thelapse of the heating time. The organic impurity compounds includediminodiacetonitrile and further included at least one compound whichexhibits a peak between 50 ppm and 90 ppm in the ¹³C-NMR spectrum and atleast one compound which exhibits absorption maximums at 380 nm and 468nm in the ultraviolet-visible absorption spectrum.

[0248] The hydrolytic activity of a microorganism to produce glycinefrom an aqueous glycinonitrile solution was evaluated with respect toeach of the above-obtained 11 samples of heated, aqueous glycinonitrilesolution and in one sample of a non-heated, aqueous glycinonitrilesolution (that is, 12 samples in total), and a comparison was made amongthe 12 samples with respect to the exhibited hydrolytic activity. Theprocedure was as follows. Using a nitrogen box, 5.0 ml of each of theglycinonitrile solution samples was individually charged into a testtube, and 5.0 ml of distilled water was added thereto. Next, usingAcinetobacter sp. AK226, a microbial suspension containing themicroorganism in an amount of 12.5% by weight in terms of the dry weightof the microorganism was prepared in the same manner as in Example 1.0.5 ml of the prepared microbial suspension was added to each test tubeand the test tubes were hermetically sealed using Parafilm. Thehermetically sealed test tubes were set in a thermostatic shaker placedin a nitrogen box, and a hydrolysis reaction was performed at 30° C. for8 hours, thereby obtaining a reaction mixture in each test tube. 2 g ofthe reaction mixture was taken out and analyzed by liquidchromatography. The results are shown in Table 1. As shown in Table 1,it was found that the yield of glycine depends on the concentration ofthe organic impurity compounds, and when the concentration of theorganic impurity compounds exceed 10% by weight, the yield of glycinedecreases markedly (see the results of Samples 11 and 12). Almost allmatter other than glycine in the reaction mixture was unreactedglycinonitrile, and from acted glycinonitrile, and from this fact, it isconsidered that the organic impurity compounds had markedly inhibitedthe activity of the microbial enzyme. On the other hand, when theconcentration of the organic impurity compounds was 5% by weight orless, the yield of glycine was 90% or more, and when the concentrationof the organic impurity compounds was 1% by weight or less, the yield ofglycine was the same as that obtained in the case of the non-heatedglycinonitrile solution. TABLE 1 Concentration of organic Heatingimpurity Sample time compounds Yield of No. (hr) (% by weight) glycine 10.0 0.5% 98% 2 0.25 1.0% 98% 3 0.5 1.5% 96% 4 1.0 2.0% 96% 5 1.5 3.0%90% 6 2.0 5.0% 91% 7 2.5 6.5% 84% 8 3.0 7.0% 80% 9 3.5 9.0% 79% 10 4.010.0% 70% 11 4.5 13.0% 13% 12 5.0 18.0%  4%

EXAMPLE 2

[0249] (1) Synthesis of Glycinonitrile

[0250] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0251] (2) Culturing of a Microorganism

[0252] Acinetobacter sp. AK226 was cultured in the same manner as inExample 1.

[0253] (3) Hydrolysis of Glycinonitrile

[0254] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 5.0% by weight in terms of the dry weight of themicroorganism. The inside of a pressure resistant Schlenk's tube used asa reactor was purged with nitrogen gas, and 1.0 ml of the microbialsuspension and 16 ml of distilled water were added thereto under a flowof nitrogen gas, followed by addition of 3 ml of the aqueousglycinonitrile solution. Then, the pressure resistant Schlenk's tube washermetically sealed and a reaction was initiated at 20° C. At this time,the pH value of the reaction system in the pressure resistant Schlenk'stube was approximately 7. Two hours after the start of the reaction, thepH value became 10.1. A part of the reaction system was taken out andanalyzed by liquid chromatography. The results of the liquidchromatography showed that glycinonitrile used as a raw material haddisappeared from the reaction system and glycine was stoichiometricallyproduced. Based on the above results, an operation which compriseselevating the reaction temperature by 5° C. and simultaneously adding 3ml of the 30% by weight aqueous glycinonitrile solution (enzymesubstrate) to the reactor was repeated every 2 hours during the reactionuntil the operation was performed 4 times. The reaction was performedfor 10 hours in total, thereby obtaining a reaction mixture in an amountof 32 ml. 2 ml of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had completely disappeared fromthe reaction system and the yield of glycine was 100%. Ammonia wasstoichiometrically produced. The amount of glycine produced per gram(dry weight) of the microorganism was 120 g/g and the activity forglycine production was 12 g/g·Hr.

[0255] The remaining 30 ml of the reaction mixture was centrifuged whilecooling to obtain a supernatant, and the obtained supernatant wassubjected to ultrafiltration, thereby obtaining a filtrate. The obtainedfiltrate was concentrated using a rotary evaporator to thereby obtain aconcentrated glycine solution. The concentrated glycine solution wascooled to thereby separate glycine by crystal-deposition. The crystalswere recovered by filtration and then dried, thereby obtaining 4.6 g ofglycine crystals. The purity of the obtained glycine was 99.99%.

EXAMPLE 3

[0256] (1) Synthesis of Glycinonitrile

[0257] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0258] (2) Culturing of a Microorganism

[0259] Acinetobacter sp. AK226 was cultured in the same manner as inExample 1.

[0260] (3) Hydrolysis of Glycinonitrile

[0261] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 12.5% by weight in terms of the dry weight of themicroorganism. The inside of a 200 ml glass autoclave was purged withnitrogen gas, and 53.3 g of the 30% by weight aqueous glycinonitrilesolution synthesized in step (1) above and 41.7 g of distilled waterwere added thereto under a nitrogen atmosphere. Subsequently, 5 g of themicrobial suspension was added to the autoclave, and a reaction wasinitiated at 50° C. At this time, the pH value of the reaction system inthe autoclave was approximately 7. After the start of the reaction, thepH value of the reaction system began to increase. One hour after thestart of the reaction, the pH value became 10. The reaction wasperformed for 8 hours, thereby obtaining a reaction mixture in an amountof 100 g. 2 g of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had completely disappeared fromthe reaction system and the yield of glycine was 99%. Ammonia wasstoichiometrically produced. The amount of glycine produced per gram(dry weight) of the microorganism was 168 g/g and the activity forglycine production was 21 g/g·Hr.

[0262] The reaction mixture was subjected to centrifugation,ultrafiltration and thin film distillation in the same manner as inExample 1, thereby obtaining 48 g of aqueous ammonia and 43 g of aconcentrated glycine solution. Glycine contained in the concentratedglycine solution was crystallized and dried, thereby obtaining 17 g ofglycine crystals. The purity of the obtained glycine was 99.99%.

EXAMPLE 4

[0263] The cells of the microorganism recovered by centrifugation inExample 3 were washed three times with distilled water. Then, distilledwater was added to the washed microorganism so as to obtain 5 g of amicrobial suspension. By using the obtained microbial suspension,glycine was produced in substantially the same manner as in Example 3.Specifically, the inside of a 200 ml glass autoclave was purged withnitrogen gas, and 50.0 g of a 30% by weight aqueous glycinonitrilesolution synthesized in the same manner as in step (1) of Example 1 and45.0 g of distilled water were added thereto under a nitrogenatmosphere. Subsequently, 5 g of the microbial suspension was added tothe autoclave, and a reaction was initiated at 50° C. The reaction wasperformed for 8 hours, thereby obtaining a reaction mixture in an amountof 100 g. 2 g of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had completely disappeared fromthe reaction system and the yield of glycine was 99%. Ammonia wasstoichiometrically produced. The amount of glycine produced per gram(dry weight) of the microorganism was 157 g/g and the activity forglycine production was 27 g/g·Hr. The sum of amounts of glycine productsproduced per gram (dry weight) of the microorganism in Examples 3 and 4was 335 g/g and the overall activity for glycine production was 20g/g·Hr.

[0264] The remainder of the reaction mixture was subjected tocentrifugation, ultrafiltration and thin film distillation in the samemanner as in Example 1, thereby obtaining 48 g of aqueous ammonia and 42g of a concentrated glycine solution. Glycine contained in theconcentrated glycine solution was crystallized and dried, therebyobtaining 16 g of glycine crystals. The purity of the obtained glycinewas 99.99%.

EXAMPLE 5

[0265] The cells of the microorganism recovered by centrifugation inExample 4 were washed three times with distilled water. Then, distilledwater was added to the washed microorganism so as to obtain 5 g of amicrobial suspension. By using the obtained microbial suspension,glycine was produced in substantially the same manner as in Example 3.Specifically, the inside of a 200 ml glass autoclave was purged withnitrogen gas, and 43.3 g of a 30% by weight aqueous glycinonitrilesolution synthesized in the same manner as in step (1) of Example 1 and51.7 g of distilled water were added thereto under a nitrogenatmosphere. Subsequently, 5 g of the microbial suspension was added tothe autoclave, and a reaction was initiated at 50° C. The reaction wasperformed for 8 hours, thereby obtaining a reaction mixture in an amountof 100 g. 2 g of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had completely disappeared fromthe reaction system and the yield of glycine was 99%. Ammonia wasstoichiometrically produced. The amount of glycine produced per gram(dry weight) of the microorganism was 136 g/g and the activity forglycine production was 17 g/g·Hr. The sum of amounts of glycine productsproduced per gram (dry weight) of the microorganism in Examples 3, 4 and5 was 461 g/g and the overall activity for glycine production was 26g/g·Hr.

EXAMPLE 6

[0266] (1) Synthesis of Glycinonitrile

[0267] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0268] (2) Culturing of a Microorganism

[0269]Rodococcus maris BP-479-9 (FERM BP-5219) was cultured at 30° C.for 1 day in a medium having the following composition.

[0270] Medium

[0271] (The medium was prepared by dissolving the below-mentionedcomponents in distilled water, and had a pH value of 7.5) glycerin 1.0%by weight meat extract 1.0 peptone 1.0 sodium chloride 0.1 ε-caprolactam0.3 potassium(I) phosphate 0.2 magnesium sulfate heptahydrate 0.02ammonium chloride 0.1 iron(II) sulfate heptahydrate 0.003 manganesechloride tetrahydrate 0.002 cobalt chloride hexahydrate 0.002

[0272] (3) Hydrolysis of Glycinonitrile

[0273] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 6.0% by weight in terms of the dry weight of themicroorganism. By using 1.0 ml of the obtained microbial suspension,glycine was produced in substantially the same manner as in Example 2.Specifically, the hydrolysis reaction of glycinonitrile was performedfor 10 hours, thereby obtaining a reaction mixture in an amount of 32 g.2 g of the reaction mixture was taken out and analyzed in the samemanner as in Example 1. As a result, it was found that glycinonitrileused as a raw material had completely disappeared from the reactionsystem and the yield of glycine was 100%. Ammonia was stoichiometricallyproduced. The amount of glycine produced per gram (dry weight) of themicroorganism was 100 g/g and the activity for glycine production was10.0 g/g·Hr.

[0274] The remainder of the reaction mixture was treated in the samemanner as in Example 1, thereby obtaining 4.6 g of glycine crystals.

EXAMPLE 7

[0275] (1) Synthesis of Glycinonitrile

[0276] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0277] (2) Culturing of a Microorganism

[0278]Corynebacterium nitrilophilus (ATCC 21419) was cultured in thesame manner as in Example 6.

[0279] (3) Hydrolysis of Glycinonitrile

[0280] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 8.0% by weight in terms of the dry weight of themicroorganism. By using 1.0 ml of the obtained microbial suspension,glycine was produced in substantially the same manner as in Example 2.Specifically, the hydrolysis reaction of glycinonitrile was performedfor 10 hours, thereby obtaining a reaction mixture in an amount of 32 g.2 g of the reaction mixture was taken out and analyzed in the samemanner as in Example 1. As a result, it was found that glycinonitrileused as a raw material had completely disappeared from the reactionsystem and the yield of glycine was 99%. Ammonia was stoichiometricallyproduced. The amount of glycine produced per gram (dry weight) of themicroorganism was 75 g/g and the activity for glycine production was 8g/g·Hr.

[0281] The remainder of the reaction mixture was treated in the samemanner as in Example 1, thereby obtaining 4.6 g of glycine crystals.

EXAMPLE 8

[0282] (1) Synthesis of Glycinonitrile

[0283] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0284] (2) Culturing of a Microorganism

[0285] Corynebacterium sp. C5 was cultured in the same manner as inExample 6.

[0286] (3) Hydrolysis of Glycinonitrile

[0287] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 12.5% by weight in terms of the dry weight of themicroorganism. There was provided a reactor comprised of a 1,000 mlseparable three-necked flask which was equipped with an agitator, ajacket for maintaining a constant temperature, a nitrogen gasintroduction nozzle which extends to the bottom of the separable flask,a mist separator connected to a dry ice trap, a thermometer and a tubefor sampling a reaction mixture. 9 ml of the microbial suspension wascharged into the separable flask and, then, 30 ml of the 30% by weightaqueous glycinonitrile solution and 161 ml of distilled water were addedthereto. A reaction was initiated at 30° C., and the reaction wasperformed while feeding nitrogen gas into the separable flask at a rateof 3 liters/hour, using a gas flow meter. 1 hour after the start of thereaction, 30 ml of the 30% by weight aqueous glycinonitrile solution wasfurther added to the separable flask. The addition of the aqueousglycinonitrile solution was performed every 1 hour 3 more times, and thereaction was conducted for 5 hours in total, thereby obtaining areaction mixture and by-products. The by-products were recovered in thedry ice trap, and when the ice and liquid recovered in the dry ice trapwere combined together, 15 g of the by-products was obtained. Theobtained by-products were dissolved in 50 ml of water, and with respectto the resultant solution, the amount of ammonia was determined by theNessler's method. It was found that 14 g of ammonia had been recoveredin the dry ice trap. The reaction mixture was obtained in an amount of305 g. 2 g of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had disappeared from the reactionsystem and the yield of glycine was 99%. Further, a trace amount ofammonia had remained in the reaction mixture. The amount of glycineproduced per gram (dry weight) of the microorganism was 53 g/g and theactivity for glycine production was 11 g/g·Hr.

[0288] The remaining 303 g of the reaction mixture was treated in thesame manner as in Example 1, thereby obtaining 47 g of glycine crystals.

EXAMPLE 9

[0289] (1) Synthesis of Glycinonitrile

[0290] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0291] (2) Culturing of a Microorganism

[0292]Alcaligenes faecalis IFO 13111 (FERM BP-4750) was cultured in thesame manner as in Example 6.

[0293] (3) Hydrolysis of Glycinonitrile

[0294] The cells of the cultured microorganism were collected from theculture broth by centrifugation and washed three times with distilledwater. Then, distilled water was added to the washed microorganism so asto obtain a microbial suspension containing the microorganism in anamount of 12.5% by weight in terms of the dry weight of themicroorganism. There was provided a reactor comprised of a 1,000 mlseparable three-necked flask which was equipped with an agitator, ajacket for maintaining a constant temperature, a nitrogen gasintroduction nozzle which extends to the bottom of the separable flask,a single column distillation tower which is connected to a vacuum pumpthrough a dry ice trap, a pressure sensor, a thermometer and a tube forsampling a reaction mixture wherein the tube is connected to a liquidtransfer pump. 11 ml of the microbial suspension was charged into theseparable flask and, then, 30 ml of the 30% by weight aqueousglycinonitrile solution and 159 ml of distilled water were addedthereto. A reaction was initiated at 30° C., and the reaction wasperformed while feeding nitrogen gas into the separable flask at a rateof 3 liters/hour, using a gas flow meter. 1 hour after the start of thereaction, 30 ml of the 30% by weight aqueous glycinonitrile solution wasfurther added to the separable flask. The addition of the aqueousglycinonitrile solution was performed every 1 hour 3 more times, and thereaction was conducted for 5 hours in total, thereby obtaining areaction mixture and by-products. The by-products were recovered in thedry ice trap, and when the ice and liquid recovered in the dry ice trapwere combined together, 25 g of the by-products were obtained. Theobtained by-products were dissolved in 50 ml of water, and with respectto the resultant solution, the amount of ammonia was determined by theNessler's method. It was found that 15 g of ammonia had been recoveredin the dry ice trap. The reaction mixture was obtained in an amount of295 g. 2 g of the reaction mixture was taken out and analyzed in thesame manner as in Example 1. As a result, it was found thatglycinonitrile used as a raw material had disappeared from the reactionsystem and the yield of glycine was 99%. Further, a trace amount ofammonia had remained in the reaction mixture. The amount of glycineproduced per gram (dry weight) of the microorganism was 42 g/g and theactivity for glycine production was 9 g/g·Hr.

[0295] The remaining 293 g of the reaction mixture was treated in thesame manner as in Example 1, thereby obtaining 48 g of glycine crystals.

EXAMPLE 10

[0296] (1) Synthesis of Glycinonitrile

[0297] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0298] (2) Culturing of a Microorganism

[0299] The below-mentioned microorganisms were individually cultured inthe same manner as in Example 6.

[0300] Microorganisms

[0301] Acinetobacter sp. AK227 (FERM BR-7413)

[0302] Mycobacterium sp. AC777 (PERM BR-2352)

[0303]Rhodopseudomonas spheroides (ATCC 11167)

[0304]Candida tropicalis (ATCC 20311)

[0305] Pseudomonas sp. 88-SB-CN5

[0306] Acremonium sp. D9K

[0307] Klebsiella sp. D5B

[0308] (3) Hydrolysis of Glycinonitrile

[0309] With resect to each of the cultured microorganisms, the followingprocedure was individually performed. The cells of the culturedmicroorganism were collected from the culture broth by centrifugationand washed three times with distilled water. Then, distilled water wasadded to the washed microorganism so as to obtain a microbial suspensioncontaining the microorganism in an amount of 12.5% by weight in terms ofthe dry weight of the microorganism. By using the obtained microbialsuspension, glycine was produced in the following manner. The inside ofa 200 ml glass autoclave was purged with nitrogen gas, and 53.3 g of the30% by weight aqueous glycinonitrile solution synthesized in step (1)above and 42.7 g of distilled water were added thereto under a nitrogenatmosphere. Subsequently, 4.0 g of the microbial suspension was added tothe autoclave, and a reaction was initiated at 40° C. The reaction wasperformed for 8 hours, thereby obtaining a reaction mixture in an amountof 100 g. 2 g of the reaction mixture was taken out. With respect to thetaken-out reaction mixture, the amounts of glycinonitrile as a rawmaterial and glycine produced were determined by liquid chromatography,and the amount of ammonia was determined by the Nessler's method. Theresults are shown in Table 2. As shown in Table 2, in the case of any ofthe employed microorganisms, glycinonitrile used as a raw material hadcompletely disappeared from the reaction system, and glycine wasproduced in high yield. Further, ammonia was stoichiometricallyproduced. TABLE 2 Conversion of glycino- Yield of nitrile glycineMicrobial strain used (%) (%) Acinetobacter sp. AK227 100 100Mycobacterium sp. AC777 100 99 Rhodopseudomonas spheroides 100 97Candida tropicalis 100 97 Pseudomonas sp. 88-SB-CN5 100 100 Acremoniumsp. D9K 100 97 Kiebsiella sp. D5B 100 97

EXAMPLE 11

[0310] (1) Synthesis of Glycinonitrile

[0311] A 30% by weight aqueous glycinonitrile solution was synthesizedin the same manner as in Example 1.

[0312] The absorbance of the aqueous glycinonitrile solution at 468 nmwas determined. It was found that the absorbance, in terms of a value asmeasured using a 10 mm quartz cell and at a concentration of 1 mol ofglycinonitrile per liter, was 0.08.

[0313] (2) Culturing of a Microorganism

[0314] The below-mentioned microorganisms were individually cultured inthe same manner as in Example 6.

[0315] Microorganisms

[0316] Acinetobacter sp. AK226 (FERM BP-2451)

[0317]Rhodococcus maris BP-479-9 (FERM BP-5219)

[0318] Corynebacterium sp. C5 (FERM BP-7414)

[0319]Corynebacterium nitrilophilus (ATCC 21419)

[0320]Alcaligenes faecalis IFO 13111 (ATCC 8750)

[0321] (3) Hydrolysis of Glycinonitrile

[0322] With resect to each of the cultured microorganisms, the followingprocedure was individually performed. The cells of the culturedmicroorganisms were collected from the culture broth by centrifugationand washed three times with distilled water. Then, distilled water wasadded to the washed microorganism so as to obtain a microbial suspensioncontaining the microorganism in an amount of 12.5% by weight in terms ofthe dry weight of the microorganism. By using the obtained microbialsuspension and a reductive compound shown in Table 3, glycine wasproduced in the following manner. The inside of a 200 ml glass autoclavewas purged with nitrogen gas, and 53.3 g of the 30% by weight aqueousglycinonitrile solution synthesized in step (1) above and 42.7 g ofdistilled water were added thereto under a nitrogen atmosphere, followedby addition of a reductive compound shown in Table 3. Then, themicrobial suspension was added to the autoclave in an amount (weight)shown in Table 3. When the amount of the microbial suspension used wasless than 5 g, distilled water was also added to the autoclave in anamount such that the total weight of the microbial suspension and thedistilled water became 5 g. A reaction was initiated at 30° C. andperformed for 10 hours. With respect to each of the microorganisms used,for comparison, an experiment was also performed which did not use areductive compound. Further, with respect to Acinetobacter sp. AK226, inorder to make a comparison between the effects obtained by using an openreaction system and the effects obtained by using a closed reactionsystem, an experiment was also conducted using an open reaction systemand not using a reductive compound. 100 g of a reaction mixture wasobtained in each experiment and 2 g of the reaction mixture was takenout. With respect to the taken-out reaction mixture, the amount ofammonia was determined by the Nessler's method, and the amounts ofglycinonitrile as a raw material and glycine produced were determined byliquid chromatography.

[0323] The remainder of the reaction mixture was subjected torefrigerating centrifugation and ultrafiltration in the same manner asin Example 1 to thereby obtain a glycine solution. A portion of theobtained glycine solution was taken out and subjected to the measurementof the absorbance at 468 nm. Glycine crystals were recovered from theremaining glycine solution by conducting thin film distillation,crystal-deposition (once), and drying. The purity of the recoveredglycine was determined. The results are shown in Table 3.

[0324] Discoloration of glycine was greatly suppressed by the additionof a reductive compound. Further, the yield of glycine was greatlyimproved and the purity of glycine crystals was 99.99%. On the otherhand, when the reaction was conducted in an open reaction system, abrown discolored reaction mixture was obtained and the yield of glycinewas lowered. In the case of the brown discolored reaction mixture,ultrafiltration of the reaction mixture was difficult, and the obtainedglycine crystals were discolored and the purity thereof was lowered to99%. TABLE 3 Microbial Yield Purity suspen- Amount Absorption Absorptionof of sion added before after glycine glycine Microorganism (g)Reductive compound (mg) reaction reaction* (%) (%) Acinetobacter 2.4Reaction in an 0 0.08 3.40 90 99.00 sp. AK226 open reaction system None0 0.08 0.79 97 99.99 L-ascorbic acid 101 0.08 0.10 100 99.99 Diammoniumsulfite 43 0.08 0.10 100 99.99 monohydrate Rhodococcus maris 3.0 None 00.08 0.79 97 99.99 BP-479-9 Formic acid 53 0.08 0.12 100 99.99 Sodiumsulfite 36 0.08 0.19 99 99.99 Corynebacterium 4.0 None 0 0.08 0.11 10099.99 nitrilophilus L-cysteine 70 0.08 0.11 100 99.99 Methylformate 690.08 0.33 100 99.99 Corynebacterium 5.0 None 0 0.08 0.79 96 99.99 sp. C5Flutatione 175 0.08 0.11 100 99.99 Ammonium formate 72 0.08 0.14 10099.99 Potassium sulfite 37 0.08 0.33 99 99.99 Alcaligenes 6.25 None 00.08 0.79 95 99.99 faecalis L-cysteine 100 0.08 0.11 100 99.99hydrochloride monohydrate Ethyl formate 85 0.08 0.19 100 99.99

EXAMPLE 12

[0325] Continuous production of glycine is explained below in detailwith reference to the production system shown in FIG. 1.

[0326] (1) Synthesis of Glycinonitrile

[0327] 1-Liter stainless steel autoclaves 6, 7 and 8 which areindividually equipped with an agitator, a level sensor, a pressuregauge, a safety valve and a temperature indicator, and flashdistillation apparatus 9 were connected with each other in series.Glycinonitrile was synthesized in the following manner. Beforeconducting the reaction, the inside of autoclaves 6, 7 and 8 and theconduits connecting the autoclaves with each other was purged withnitrogen gas. Three diaphragm pumps were provided and the diaphragmpumps were respectively used for continuously feeding 37% by weightaqueous formaldehyde solution 1 (containing methyl alcohol in an amountof less than 10% by weight) at a feeding rate of 100 g/hr (the aqueousformaldehyde solution 1 had previously been treated for replacing oxygendissolved therein by nitrogen), liquid hydrogen cyanide 2 at a feedingrate of 33.3 g/hr, and distilled water containing 100 ppm of 4% sodiumhydroxide 3 at a feeding rate of 240 g/hr into autoclave 6. The reactiontemperature was set at 40° C. and the reaction for producingglycolonitrile was initiated. Generation of heat was observedimmediately after the start of the reaction, but thereafter the reactionproceeded at the set reaction temperature. Residence time was adjustedto 1 hour by using the level controller, and a reaction mixturecontaining glycolonitrile was fed from autoclave 7 into autoclave 8 byusing a pump, while continuously feeding ammonia gas 4 under a pressureof 0.2 MPa into autoclave 7 at a feeding rate of 82 normal liters/hrthrough a mass flow controller. The reaction temperature of autoclave 7was set at 55° C. and the reaction for producing glycinonitrile wasinitiated. Generation of heat due to the absorption of ammonia wasobserved at the start of the reaction, but thereafter the reactionproceeded at the set reaction temperature and pressure. The residencetime in autoclave 7 was adjusted to 1 hour by using a level controller,and a reaction mixture was fed from autoclave 7 into autoclave 8 byusing a pump. The temperature of autoclave 8 was set at 55° C. After theresidence time of 1 hour passed, a reaction mixture containingglycinonitrile was continuously withdrawn from autoclave 8 by using apump. A primary reaction mixture obtained during the first 6 hours fromthe start of the reaction was collected as a waste and the reactionmixture obtain after 6 hours from the start of the reaction and obtainedlater was fed into flash distillation apparatus 9 set at the atmosphericpressure. The column top temperature of flash distillation apparatus 9was set at −20° C., and the reaction mixture was cooled at the columntop of distillation apparatus 9. Moisture contained in the reactionmixture was removed by cooling the reaction mixture with ice, and moistammonia gas was recovered by a dry ice-ethanol trap, thereby obtainingliquid ammonia 10. Recovered liquid ammonia 10 was used as a rawmaterial to be fed into autoclave 7. An aqueous glycinonitrile solutioncontaining a small amount of ammonia was recovered from the columnbottom of the flash distillation apparatus, and the recoveredglycinonitrile solution was stored at 5° C. in intermediate tank 11. Forthe storage, 100 ppm of 10% by weight sulfuric acid as a stabilizer wasadded to the glycinonitrile solution in intermediate tank 11. A 33% byweight glycinonitrile solution was obtained. The obtained glycinonitrilesolution contained 0.9% by weight of iminodiacetonitrile and 1.3% byweight of at least one organic impurity compound having a molecularweight of 95 or more.

[0328] (2) Culturing of a Microorganism

[0329] Acinetobacter sp. AK226 (FERM BP-2451) was cultured in the samemanner as in Example 1. The cells of the cultured microorganism werecollected from the culture broth by centrifugation and washed threetimes with distilled water. Then, distilled water was added to thewashed microorganism so as to obtain a microbial suspension 5 containingthe microorganism in an amount of 12.5% by weight in terms of the dryweight of the microorganism.

[0330] (3) Hydrolysis Reaction and Separation of a Microorganism

[0331] A 10-liter autoclave equipped with an agitator, a thermometer anda pH sensor was used as hydrolysis reactor 12. 5,400 g of the aqueousglycinonitrile solution stored in intermediate tank 11 was charged intohydrolysis reactor 12 under a flow of nitrogen gas and then, 60 g ofmicrobial suspension 5 prepared in step (2) above was added thereto. Thehydrolysis reaction of glycinonitrile was initiated at a set reactiontemperature of 50° C. After conducting the reaction for 8 hours, theresultant reaction mixture was transferred into continuous centrifuge 13(model TA1-02, manufactured and sold by Westfalia Separator Inc.,U.S.A.), wherein the microorganism was separated from the reactionmixture by centrifugal filtration. One fifth of the separatedmicroorganism (microorganism slurry) was discarded (microorganism to bediscarded 14) and the remainder of the microorganism was washed threetimes with distilled water. Then, distilled water was added to thewashed microorganism so as to obtain 48 g of a recovered microbialsuspension. 12 g of a microbial suspension prepared in the same manneras in step (2) above was mixed with the recovered microbial suspension,thereby obtaining 60 g of a microbial suspension. The obtained 60 g ofmicrobial suspension was charged into hydrolysis reactor 12, and ahydrolysis of glycinonitrile was conducted therein in the same manner asmentioned above. In this way, the reaction operation was repeatedincluding the above-described operation for separating and recycling themicroorganism. The filtrate obtained by the centrifugal filtration(i.e., the reaction mixture containing glycine) was transferred tocirculation type ultrafiltration apparatus 15 (using ultrafiltrationmembrane: “SIP-1013”, manufactured and sold by Asahi Kasei KabushikiKaisha, Japan) and concentrated therein while being circulated. Theresultant concentrated reaction mixture was transferred to intermediatetank 16 and stored therein. When the volume of the circulating liquiddecreased to 500 ml, 500 ml of distilled water was added thereto so thatthe total volume of the mixture became 1,000 ml, and the mixture wasconcentrated again until the volume of the circulating liquid became 500ml. This cycle comprising diluting the liquid and then concentrating thediluted liquid in the ultrafiltration apparatus was performed twice, andthen 500 ml of the circulating liquid remaining in ultrafiltrationapparatus 15 was discarded.

[0332] Step (3) above for producing glycine was repeated to obtain theresults shown below, wherein one cycle of step (3) took 12 hours.

[0333] The concentrated reaction mixture contained 83% by weight ofammonium salt of glycine and 0.9% by weight of at least one organicimpurity compound having a molecular weight of 95 or more.

[0334] (4) Recovery of Glycine Crystals

[0335] After 18 hours from the start of the reaction in step (1) above,approximately 6 kg of the concentrated reaction mixture was obtained inintermediate tank 16. Then, continuous crystal-deposition of glycine wasinitiated using the concentrated reaction mixture in intermediate tank16. The procedure is as described below. The concentrated reactionmixture was flowed through activated carbon column 17, thereby obtaininga concentrate which had been treated with an activated carbon. Theobtained activated carbon-treated concentrate was fed into continuouscrystal-deposition apparatus 18 at a flow rate of 425 g/hr forperforming a crystal-deposition operation. Continuous crystal-depositionapparatus 18 was equipped with an agitator, a level sensor, athermometer and a vacuum distillation column. Excess water and liquidammonia contained in the concentrate were removed therefrom by vacuumevaporation at rates of approximately 200 g/hr and approximately 20g/hr, respectively. The evaporated water and liquid ammonia were,respectively, recovered as distilled water 19 and liquid ammonia 10, andboth distilled water 19 and liquid ammonia 10 were recycled for use inthe above-mentioned step (1). A slurry (glycine concentration: 40% byweight) was accumulated in a crystal-deposition vessel. A part of theslurry accumulated in the crystal-deposition vessel was intermittentlywithdrawn from the bottom of the crystal-deposition vessel by suction sothat the surface of the slurry in the crystal-deposition vessel wasmaintained between the predetermined upper and lower levels in thevessel. The withdrawn slurry was subjected to filtration while heatingto obtain a filtrate. The obtained filtrate was blown down (blow 20)until the weight thereof exhibited a 4% decrease, to thereby obtainfiltrate 21, and filtrate 21 was recycled to the crystal-depositionvessel. The crystal-deposition operation reached a stationary stateafter 3 hours from the start of the crystal-deposition operation andglycine crystals were deposited in the crystal-deposition vessel. Thedeposited glycine crystals were taken out from crystal-depositionapparatus 18 and dried to thereby obtain glycine crystals 22. The purityof glycine crystals 22 was determined to be 99.99%. In this productionsystem, glycine was produced at an average rate of 82 g/hr (54 g/g ofthe microorganism), and the overall yield of glycine was 90% (whereinthe overall yield (%) is a value calculated by subtracting all losses(%) in all steps).

INDUSTRIAL APPLICABILITY

[0336] By the use of the method of the present invention, a high purityglycine which is useful as a food additive and as a raw material forsynthesizing pharmaceuticals, agricultural chemicals and detergents canbe produced while achieving advantages in that both the glycineproduction activity per unit weight of a microorganism and the glycineproduction activity per unit time become high, and both glycine andammonia can be stoichiometrically produced without decomposition orconsumption thereof and can be separately and easily recovered. Further,glycine can be produced on a commercial scale without causing a heavyburden on the environment.

1. A method for producing glycine, comprising: providing glycinonitrilein the form of an aqueous solution thereof, subjecting the aqueoussolution of glycinonitrile to a hydrolysis reaction in a hydrolysisreaction system under the action of a microbial enzyme having theactivity to hydrolyze a nitrile group, thereby converting saidglycinonitrile to glycine while by-producing ammonia, said hydrolysisreaction system containing at least one organic impurity compoundinhibiting said microbial enzyme, wherein said at least one organicimpurity compound inhibiting said microbial enzyme has a molecularweight of 95 or more and contains at least one member selected from thegroup consisting of a nitrile group, a carboxyl group, an amide group,an amino group, a hydroxyl group and a trimethyleneamine structure,wherein said trimethyleneamine structure has a skeleton represented bythe following formula (1):

wherein n represents an integer of 1 or more, said hydrolysis reactionbeing performed under conditions wherein, during said hydrolysisreaction, the content of said organic impurity compound inhibiting saidmicrobial enzyme in said hydrolysis reaction system is maintained at alevel of 10% by weight or less, based on the weight of said hydrolysisreaction system, and isolating the glycine from the hydrolysis reactionsystem.
 2. The method according to claim 1, wherein said at least oneorganic impurity compound inhibiting said microbial enzyme is producedas a by-product in at least one reaction selected from the groupconsisting of the synthesis of glycinonitrile from hydrogen cyanide,formaldehyde and ammonia, and the hydrolysis of the glycinonitrile intoglycine and ammonia.
 3. The method according to claim 1 or 2, whereinsaid at least one organic impurity compound inhibiting said microbialenzyme comprises a compound represented by the following formula (2):NH_(3-n)(CH_(Z)Y¹)_(n)  (2) wherein each Y¹ independently represents anitrile group, a carboxyl group or an amide group; and n represents aninteger of 2 or
 3. 4. The method according to claim 1 or 2, wherein saidat least one organic impurity compound inhibiting said microbial enzymecomprises at least one compound selected from the group consisting ofthe following compounds (a) and (b): (a) a compound represented by thefollowing formula (3):

wherein: Y¹ represents a nitrile group, a carboxyl group or an amidegroup; each Y² independently represents an amino group or a hydroxylgroup; n represents an integer of 0 or more; and the or each Z isindependently represented by the following formula (4) or (5):

wherein each Y independently represents an amino group or a hydroxylgroup, and (b) a compound represented by the following formula (6) or(7):

wherein each Y² independently represents an amino group or a hydroxylgroup; and each Z² is independently represented by the following formula(8) or (9):

wherein: Y² represents an amino group or a hydroxyl group; the or eachZ¹ is as defined for formula (3); and n represents an integer of 0 ormore.
 5. The method according to claim 1 or 2, wherein said at least oneorganic impurity compound inhibiting said microbial enzyme comprises atleast one compound selected from the group consisting of the followingcompounds (c) and (d): (c) a compound represented by the followingformula (10) or (11):

wherein each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group, and (d) a compound represented by the followingformula (12): (HCN)_(n)  (12) wherein n represents an integer of 4 ormore.
 6. The method according to claim 1 or 2, wherein said at least oneorganic impurity compound inhibiting said microbial enzyme comprises acompound having in a molecule thereof at least one skeleton selectedfrom the group consisting of the following skeletons (e) and (f): (e) askeleton represented by the following formula (13):

wherein each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group; and n represents an integer of 2 or more, and(f) a skeleton represented by the following formula (14) or (15):

wherein: the or each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; each Y² independently represents anamino group or a hydroxyl group; and n represents an integer of 1 ormore.
 7. The method according to claim 1 or 2, wherein said at least oneorganic impurity compound inhibiting said microbial enzyme comprises acompound having in a molecule thereof at least one skeleton selectedfrom the group consisting of the following skeletons (g) and (h): (g) askeleton represented by the following formula (16):

wherein: each Y¹ independently represents a nitrile group, a carboxylgroup or an amide group; each Y² independently represents an amino groupor a hydroxyl group; and n represents an integer of 2 or more, and (h) askeleton represented by the following formula (17) or (18):

wherein: the or each Y¹ independently represents a nitrile group, acarboxyl group or an amide group; each Y² independently represents anamino group or a hydroxyl group; and n represents an integer of 1 ormore.
 8. The method according to claim 1 or 2, wherein said at least oneorganic impurity compound inhibiting said microbial enzyme compriseshexamethylenetetramine.
 9. The method according to any one of claims 1to 8, wherein said at least one organic impurity compound inhibitingsaid microbial enzyme exhibits a peak between 53 ppm and 100 ppm in a¹³C-NMR spectrum as measured in heavy water.
 10. The method according toany one of claims 1 to 9, wherein said at least one organic impuritycompound inhibiting said microbial enzyme exhibits absorption maximumsbetween 340 nm and 380 nm and between 440 nm and 480 nm in anultraviolet-visible absorption spectrum as measured with respect to saidhydrolysis reaction system.
 11. The method according to any one ofclaims 1 to 10, wherein said at least one organic impurity compoundinhibiting said microbial enzyme has a molecular weight of 130 or more.12. The method according to any one of claims 1 to 11, wherein theamount of said at least one organic impurity compound inhibiting saidmicrobial enzyme is 1% by weight or less, based on the weight of saidhydrolysis reaction system.
 13. The method according to any one ofclaims 1 to 12, wherein said hydrolysis reaction system has oxygendissolved therein in an amount of 5 ppm by weight or less, based on theweight of said hydrolysis reaction system.
 14. The method according toany one of claims 1 to 13, wherein said hydrolysis is conducted using aclosed reaction system, a reaction system which is pressurized with aninert gas, a reaction system through which an inert gas is flowed or areaction system which is under a pressure of less than atmosphericpressure, so that the amount of oxygen dissolved in said hydrolysisreaction system is suppressed.
 15. The method according to any one ofclaims 1 to 14, wherein said hydrolysis is conducted in said hydrolysisreaction system having ammonia dissolved therein.
 16. The methodaccording to any one of claims 1 to 15, wherein said hydrolysis isconducted in said hydrolysis reaction system containing an electrolytein an amount of 2% by weight or less, based on the weight of theglycinonitrile.
 17. The method according to any one of claims 1 to 16,wherein said microbial enzyme having the activity to hydrolyze a nitrilegroup is derived from a microorganism belonging to a genus selected fromthe group consisting of Acinetobacter, Rhodococcus, Corynebacterium,Alcaligenes, Mycobacterium, Rhodopseudomonas and Candida.
 18. The methodaccording to claim 17, wherein the microbial strain of saidAcinetobacter is Acinetobacter sp. AK226, deposited with the NationalInstitute of Bioscience and Human-Technology, Japan under the accessionnumber FERM BP-2451, or Acinetobacter sp. AK227, deposited with theNational Institute of Bioscience and Human-Technology, Japan under theaccession number FERM BP-7413.
 19. The method according to claim 17,wherein the microbial strain of said Rhodococcus is Rhodococcus marisBP-479-9, deposited with the National Institute of Bioscience andHuman-Technology, Japan under the accession number FERM BP-5219.
 20. Themethod according to claim 17, wherein the microbial strain of saidCorynebacterium is Corynebacterium sp. C5, deposited with the NationalInstitute of Bioscience and Human-Technology, Japan under the accessionnumber FERM BP-7414, or Corynebacterium nitrilophilus, deposited withthe American Type Culture Collection, U.S.A under the accession numberATCC
 21419. 21. The method according to claim 17, wherein the microbialstrain of said Alcaligenes is Alcaligenes faecalis IFO 13111, depositedwith the National Institute of Bioscience and Human-Technology, Japanunder the accession number FERM BP-4750.
 22. The method according toclaim 17, wherein the microbial strain of said Mycobacterium isMycobacterium sp. AC777, deposited with the National Institute ofBioscience and Human-Technology, Japan under the accession number FERMBP-2352.
 23. The method according to claim 17, wherein the microbialstrain of said Rhodopseudomonas is Rhodopseudomonas spheroides,deposited with the American Type Culture Collection, U.S.A under theaccession number ATCC
 11167. 24. The method according to claim 17,wherein the microbial strain of said Candida is Candida tropicalis,deposited with the American Type Culture Collection, U.S.A under theaccession number ATCC
 20311. 25. The method according to any one ofclaims 1 to 24, wherein the isolation of the glycine from saidhydrolysis reaction system is conducted while recovering the by-producedammonia separately from the recovery of glycine.
 26. The methodaccording to claim 25, wherein the glycine and the ammonia areseparately recovered by at least one operation selected from the groupconsisting of distillation, reactive distillation, entrainment by aninert gas, ion exchange, extraction, reprecipitation using a poorsolvent, and crystal-deposition by concentration or cooling.
 27. Themethod according to claim 26, wherein the ammonia is recovered bydistillation, reactive distillation or entrainment by an inert gas, andthe glycine is recovered by subjecting a liquid remaining after therecovery of the ammonia to crystal-deposition by concentration orcooling.
 28. The method according to any one of claims 1 to 27, whichcomprises: (1) reacting hydrogen cyanide with formaldehyde in an aqueousmedium in the presence of an alkali catalyst in a closed reaction systemto obtain glycolonitrile in the form of an aqueous solution thereof, (2)adding ammonia to the aqueous solution of glycolonitrile to effect areaction between the glycolonitrile and the ammonia, to thereby obtainglycinonitrile in the form of an aqueous solution thereof whileproducing water, (3) separating most of the ammonia and a part of thewater from the obtained aqueous solution of glycinonitrile bydistillation to thereby obtain a hydrolysis reaction system containingthe glycinonitrile in the form of an aqueous solution thereof and theammonia remaining unseparated, wherein the separated ammonia isrecovered for recycle thereof to step (2), (4) subjecting saidhydrolysis reaction system to a hydrolysis reaction under the action ofa microbial enzyme produced by a microorganism added in said hydrolysisreaction system which is in a closed system, thereby converting saidglycinonitrile to glycine while by-producing ammonia, (5) separatingsaid microorganism and said microbial enzyme by at least one operationselected from the group consisting of centrifugal filtration andmembrane filtration, wherein said microorganism and said microbialenzyme are recovered for recycle thereof to step (4), (6) separating apart of organic impurity compounds inhibiting said microbial enzymewhich compounds are by-produced in steps (1) to (5) by at least oneoperation selected from the group consisting of membrane filtration andadsorbent-separation, (7) separating by distillation the ammoniaby-produced in step (4) and an excess amount of water which remains insaid hydrolysis reaction system after step (4), wherein the separatedammonia is recovered for recycle thereof to step (2), (8) after orsimultaneously with step (7), separating said glycine bycrystal-deposition, and (9) drying the crystals of said glycine.
 29. Amethod for producing glycine, comprising providing glycinonitrile in theform of an aqueous solution thereof, subjecting the aqueous solution ofglycinonitrile to a hydrolysis reaction, thereby converting saidglycinonitrile to glycine while by-producing ammonia, and isolating theglycine from the hydrolysis reaction system, wherein said hydrolysis ofglycinonitrile is conducted in the presence of ammonia.
 30. The methodaccording to claim 29, wherein the amount of the ammonia is from 0.001to 5 mol, relative to 1 mole of the glycinonitrile.
 31. A method forproducing glycine, comprising providing glycinonitrile in the form of anaqueous solution thereof, subjecting the aqueous solution ofglycinonitrile to a hydrolysis reaction, thereby converting saidglycinonitrile to glycine while by-producing ammonia, and isolating theglycine from the hydrolysis reaction system, wherein the isolation ofthe glycine from said hydrolysis reaction system is conducted whilerecovering the by-produced ammonia separately from the recovery ofglycine in the absence of a base and an acid.
 32. The method accordingto claim 31, wherein the glycine and ammonia are separately recovered byat least one operation selected from the group consisting ofdistillation, reactive distillation, entrainment by an inert gas, ionexchange, extraction, reprecipitation using a poor solvent, andcrystal-deposition by concentration or cooling.
 33. The method accordingto claim 32, wherein the ammonia is recovered by distillation, reactivedistillation or entrainment by an inert gas, and the glycine isrecovered by subjecting a liquid remaining after the recovery of theammonia to crystal-deposition by concentration or cooling.